Cloned dna polymerases from thermotoga and mutants thereof

ABSTRACT

The invention relates to a substantially pure thermostable DNA polymerase from  Thermotoga  (Tne and Tma) and mutants thereof. The Tne DNA polymerase has a molecular weight of about 100 kilodaltons and is more thermostable than Taq DNA polymerase. The mutant DNA polymerase has at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3′→5′ exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5′→3′ exonuclease activity of said DNA polymerase; (3) a third mutation in the O helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides. The present invention also relates to the cloning and expression of the wild type or mutant DNA polymerases in  E. coli , to DNA molecules containing the cloned gene, and to host cells which express said genes. The DNA polymerases of the invention may be used in well-known DNA sequencing and amplification reactions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. application Ser. No. 08/______,filed Aug. 14, 1996, pending, which is a continuation-in-part of U.S.application Ser. No. 08/537,400, filed Oct. 2, 1995, pending, which is acontinuation-in-part of U.S. application Ser. No. 08/370,190, filed Jan.9, 1995, pending, which is a continuation-in-part of U.S. applicationSer. No. 08/316,423, filed Sep. 30, 1994, now abandoned. This is also acontinuation-in-part of U.S. application Ser. No. 08/576,759, filed Dec.21, 1995, which is a continuation of U.S. application Ser. No.08/537,397, filed Oct. 2, 1995, which is a continuation-in-part of U.S.application Ser. No. 08/525,057, filed Sep. 8, 1995. The contents ofeach of these applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a substantially pure thermostable DNApolymerase. Specifically, the DNA polymerase of the present invention isa Thermotoga DNA polymerase and more specifically a Thermotoganeapolitana (Tne) DNA polymerase or Thermotoga maritima (Tma) DNApolymerase. Preferably, the polymerase has a molecular weight of about100 kilodaltons. The present invention also relates to the cloning andexpression of the Thermotoga DNA polymerase in E. coli, to DNA moleculescontaining the cloned gene, and to hosts which express said genes. TheDNA polymerase of the present invention may be used in DNA sequencing,amplification reactions, and cDNA synthesis.

This invention also relates to mutants of the Thermotoga DNA polymerase,including Tne and Tma DNA polymerase. Specifically, the DNA polymerasesof the present invention have mutations which substantially reduce 3′→5′exonuclease activity; mutations resulting in the ability of the mutantDNA polymerase to incorporate dideoxynucleotides into a DNA moleculeabout as efficiently as deoxynucleotides; and mutations whichsubstantially reduce 5′→3′ exonuclease activity. The Thermotoga (e.g.,Tne and Tma) mutant DNA polymerase of this invention can have one ormore of these properties. These DNA polymerase mutants may also be usedin DNA sequencing, amplification reactions, and cDNA synthesis.

The present invention is also directed to novel mutants of other DNApolymerases which have substantially reduced 5′-3′ exonuclease activity.

2. Background Information

DNA polymerases synthesize the formation of DNA molecules which arecomplementary to a DNA template. Upon hybridization of a primer to thesingle-stranded DNA template, polymerases synthesize DNA in the 5′ to 3′direction, successively adding nucleotides to the 3′-hydroxyl group ofthe growing strand. Thus, in the presence of deoxyribonucleosidetriphosphates (dNTPs) and a primer, a new DNA molecule, complementary tothe single stranded DNA template, can be synthesized.

A number of DNA polymerases have been isolated from mesophilicmicroorganisms—such as E. coli. A number of these mesophilic DNApolymerases have also been cloned. Lin et al. cloned and expressed T4DNA polymerase in E. coli (Proc. Natl. Acad. Sci. USA 84:7000-7004(1987)). Tabor et al. (U.S. Pat. No. 4,795,699) describes a cloned T7DNA polymerase, while Minkley et al. (J. Biol. Chem. 259:10386-10392(1984)) and Chatterjee (U.S. Pat. No. 5,047,342) described E. coli DNApolymerase I and the cloning of T5 DNA polymerase, respectively.

Although DNA polymerases from thermophiles are known, relatively littleinvestigation has been done to isolate and even clone these enzymes.Chien et al., J Bacteriol. 127:1550-1557 (1976) describe a purificationscheme for obtaining a polymerase from Thermus aquaticus (Taq). Theresulting protein had a molecular weight of about 63,000 daltons by gelfiltration analysis and 68,000 daltons by sucrose gradientcentrifugation. Kaledin et al., Biokhymiya 45:644-51 (1980) disclosed apurification procedure for isolating DNA polymerase from T. aquaticusYT1 strain. The purified enzyme was reported to be a 62,000 daltonmonomeric protein. Gelfand et al. (U.S. Pat. No. 4,889,818) cloned agene encoding a thermostable DNA polymerase from Thermus aquaticus. Themolecular weight of this protein was found to be about 86,000 to 90,000daltons.

Simpson et al. purified and partially characterized a thermostable DNApolymerase from a Thermotoga species (Biochem. Cell. Biol. 86:1292-1296(1990)). The purified DNA polymerase isolated by Simpson et al.exhibited a molecular weight of 85,000 daltons as determined bySDS-polyacrylamide gel electrophoresis and size-exclusionchromatography. The enzyme exhibited half-lives of 3 minutes at 95° C.and 60 minutes at 50° C. in the absence of substrate and its pH optimumwas in the range of pH 7.5 to 8.0. Triton X-100 appeared to enhance thethermostability of this enzyme. The strain used to obtain thethermostable DNA polymerase described by Simpson et al. was Thermotogaspecies strain FjSS3-B.1 (Hussar et al., FEMS Microbiology Letters37:121-127 (1986)). Others have cloned and sequenced a thermostable DNApolymerase from Thermotoga maritima (U.S. Pat. No. 5,374,553, which isexpressly incorporated herein by reference).

Other DNA polymerases have been isolated from thermophilic bacteriaincluding Bacillus steraothermophilus (Stenesh et al., Biochim. Biophys.Acta 272:156-166 (1972); and Kaboev et al., J. Bacteriol. 145:21-26(1981)) and several archaebacterial species (Rossi et al., System. Appl.Microbiol. 7:337-341 (1986); Klimczak et al., Biochemistry 25:4850-4855(1986); and Elie et al., Eur. J. Biochem. 178:619-626 (1989)). The mostextensively purified archaebacterial DNA polymerase had a reportedhalf-life of 15 minutes at 87° C. (Elie et al. (1989), supra). Innis etal., In PCR Protocol: A Guide To Methods and Amplification, AcademicPress, Inc., San Diego (1990) noted that there are several extremethermophilic eubacteria and archaebacteria that are capable of growth atvery high temperatures (Bergquist et al., Biotech. Genet. Eng. Rev.5:199-244 (1987); and Kelly et al., Biotechnol. Prog. 4:47-62 (1988))and suggested that these organisms may contain very thermostable DNApolymerases.

In many of the known polymerases, the 5′→3′ exonuclease activity ispresent in the N-terminal region of the polymerase. (Ollis, et al.,Nature 313:762-766 (1985); Freemont et al., Proteins 1:66-73 (1986);Joyce, Cur. Opin. Struct. Biol. 1:123-129 (1991).) There are some aminoacids, the mutation of which are thought to impair the 5′→3′ exonucleaseactivity of E. coli DNA polymerase I. (Gutman & Minton, Nucl. Acids Res.21:44064407 (1993).) These amino acids include Tyr⁷⁷, Gly¹⁰³, Gly¹⁸⁴,and Gly¹⁹² in E. coli DNA polymerase I. It is known that the5′-exonuclease domain is dispensable. The best known example is theKlenow fragment of E. coli polymerase I. The Klenow fragment is anatural proteolytic fragment devoid of 5′-exonuclease activity (Joyceet. al., J. Biol. Chem. 257:1958-64 (1990).) Polymerases lacking thisactivity are useful for DNA sequencing.

Most DNA polymerases also contain a 3′→5′ exonuclease activity. Thisexonuclease activity provides a proofreading ability to the DNApolymerase. A T5 DNA polymerase that lacks 3′→5′ exonuclease activity isdisclosed in U.S. Pat. No. 5,270,179. Polymerases lacking this activityare particularly useful for DNA sequencing.

The polymerase active site, including the dNTP binding domain is usuallypresent at the carboxyl terminal region of the polymerase (Ollis et al.,Nature 313:762-766 (1985); Freemont et al., Proteins 1:66-73 (1986)). Ithas been shown that Phe⁷⁶² of E. coli polymerase I is one of the aminoacids that directly interacts with the nucleotides (Joyce & Steitz, Ann.Rev. Biochem. 63:777-822 (1994); Astalke, J. Biol. Chem. 270:1945-54(1995)). Converting this amino acid to a Tyr results in a mutant DNApolymerase that does not discriminate against dideoxynucleotides. Seecopending U.S. application Ser. No. 08/525,087, of Deb K. Chatterjee,filed Sep. 8, 1995, entitled “Mutant DNA Polymerases and the UseThereof,” which is expressly incorporated herein by reference.

Thus, there exists a need in the art to develop more thermostable DNApolymerases. There also exists a need in the art to obtain wild type ormutant DNA polymerases that are devoid of exonuclease activities and arenon-discriminating against dideoxynucleotides.

SUMMARY OF THE INVENTION

The present invention satisfies these needs in the art by providingadditional DNA polymerases useful in molecular biology. Specifically,this invention includes a thermostable DNA polymerase. Preferably, thepolymerase has a molecular weight of about 100 kilodaltons.Specifically, the DNA polymerase of the invention is isolated fromThermotoga, and more specifically, the DNA polymerase is obtained fromThermotoga neapolitana (Tne) and Thermotoga maritima (Tma). TheThermotoga species preferred for isolating the DNA polymerase of thepresent invention was isolated from an African continental solfataricspring (Windberger et al., Arch. Microbiol. 151. 506-512, (1989)).

The Thermotoga DNA polymerases of the present invention are extremelythermostable, showing more than 50% of activity after being heated for60 minutes at 90° C. with or without detergent. Thus, the DNApolymerases of the present invention is more thermostable than Taq DNApolymerase.

The present invention is also directed to cloning a gene encoding aThermotoga DNA polymerase enzyme. DNA molecules containing theThermotoga DNA polymerase genes, according to the present invention, canbe transformed and expressed in a host cell to produce the DNApolymerase. Any number of hosts may be used to express the ThermotogaDNA polymerase gene of the present invention; including prokaryotic andeukaryotic cells. Preferably, prokaryotic cells are used to express theDNA polymerase of the invention. The preferred prokaryotic hostaccording to the present invention is E. coli.

The present invention also relates mutant thermostable DNA polymerasesof the PolI type and DNA coding therefor, wherein there is amino acidchange in the O-helix which renders the polymerase nondiscriminatoryagainst ddNTPs in sequencing reactions. The O-helix is defined asRXXXKXXXFXXXYX, wherein X is any amino acid.

The present invention also relates to Thermotoga DNA polymerase mutantsthat lack exonuclease activity and/or which are nondiscriminatoryagainst ddNTPs in sequencing reactions.

The present invention is also directed generally to DNA polymerases thathave mutations that result in substantially reduced or missing 5′→3′exonuclease activity.

In particular, the invention relates to a Thermotoga DNA polymerasemutant which is modified at least one way selected from the groupconsisting of (a) to reduce or eliminate the 3′-5′ exonuclease activityof the polymerase;

(b) to reduce or eliminate the 5′-3′ exonuclease activity of thepolymerase; and

(c) to reduce or eliminate discriminatory behavior against adideoxynucleotide.

The invention also relates to a method of producing a DNA polymerase,said method comprising:

(a) culturing the host cell of the invention;

(b) expressing said gene; and

(c) isolating said DNA polymerase from said host cell.

The invention also relates to a method of synthesizing a double-strandedDNA molecule comprising:

(a) hybridizing a primer to a first DNA molecule; and

(b) incubating said DNA molecule of step (a) in the presence of one ormore deoxy- or dideoxyribonucleoside triphosphates and the DNApolymerase of the invention, under conditions sufficient to synthesize asecond DNA molecule complementary to all or a portion of said first DNAmolecule. Such deoxy- and dideoxyribonucleoside triphosphates includedATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, 7-deaza-dATP, dUTP, ddATP,ddCTP, ddGTP, ddITP, ddTTP, [α-S]dATP, [α-S]dTTP, [α-S]dGTP, and[α-S]dCTP.

The invention also relates to a method of sequencing a DNA molecule,comprising:

(a) hybridizing a primer to a first DNA molecule;

(b) contacting said DNA molecule of step (a) with deoxyribonucleosidetriphosphates, the DNA polymerase of the invention, and a terminatornucleotide;

(c) incubating the mixture of step (b) under conditions sufficient tosynthesize a random population of DNA molecules complementary to saidfirst DNA molecule, wherein said synthesized DNA molecules are shorterin length than said first DNA molecule and wherein said synthesized DNAmolecules comprise a terminator nucleotide at their 3′ termini; and

(d) separating said synthesized DNA molecules by size so that at least apart of the nucleotide sequence of said first DNA molecule can bedetermined. Such terminator nucleotides include ddTTP, ddATP, ddGTP,ddITP or ddCTP.

The invention also relates to a method for amplifying a double strandedDNA molecule, comprising:

(a) providing a first and second primer, wherein said first primer iscomplementary to a sequence at or near the 3′-termini of the firststrand of said DNA molecule and said second primer is complementary to asequence at or near the 3′-termini of the second strand of said DNAmolecule;

(b) hybridizing said first primer to said first strand and said secondprimer to said second strand in the presence of the DNA polymerase ofthe invention, under conditions such that a third DNA moleculecomplementary to said first strand and a fourth DNA moleculecomplementary to said second strand are synthesized;

(c) denaturing said first and third strand, and said second and fourthstrands; and

(d) repeating steps (a) to (c) one or more times.

The invention also relates to a kit for sequencing a DNA molecule,comprising:

(a) a first container means comprising the DNA polymerase of theinvention;

(b) a second container means comprising one or moredideoxyribonucleoside triphosphates; and

(c) a third container means comprising one or more deoxyribonucleosidetriphosphates.

The invention also relates to a kit for amplifying a DNA molecule,comprising:

(a) a first container means comprising the DNA polymerase of theinvention; and

(b) a second container means comprising one or more deoxyribonucleosidetriphosphates.

The present invention also relates to a mutant DNA polymerase havingsubstantially-reduced or eliminated 5′-3′ exonuclease activity, whereinat least one of the amino acids corresponding to Asp⁸, Glu¹¹², Asp¹¹⁴,Asp¹¹⁴, Asp¹¹⁵, Asp¹³⁷, Asp¹³⁹, Gly¹⁰², Gly¹⁸⁷, or Gly¹⁹⁵ of Tne DNApolymerase has been mutated.

The present invention also relates to a method of producing a mutant DNApolymerase having substantially reduced or eliminated 5′-3′ exonucleaseactivity, wherein at least one of the amino acids corresponding to Asp⁸,Glu¹¹², Asp¹¹⁴, Asp¹¹⁵, Asp¹³⁷, Asp¹³⁹, Gly¹⁰² Gly¹⁸⁷, or Gly¹⁹⁵ of TneDNA polymerase has been mutated, comprising:

(a) culturing the host cell of the invention;

(b) expressing the mutant DNA polymerase; and

(c) isolating said mutant DNA polymerase.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates the heat stability of Tne DNA polymerase at 90° C.over time. Partially purified DNA polymerase from the crude extract ofThermotoga neapolitana cells was used in the assay.

FIG. 2 shows the time-dependent DNA polymerase activity of Tne DNApolymerase isolated from an E. coli host containing the cloned Tne DNApolymerase gene.

FIG. 3 compares the ability of various DNA polymerases to incorporateradioactive dATP and [αS]dATP. Tne DNA polymerase is more effective atincorporating [αS]dATP than was Taq DNA polymerase.

FIG. 4 shows the restriction map of the approximate DNA fragment whichcontains the Tne DNA polymerase gene in pSport 1 and pUC19. This figurealso shows the region containing the O-helix homologous sequences.

FIGS. 5A and 5B shows the nucleotide and deduced amino acid sequences,in all 3 reading frames, for the carboxyl terminal portion, includingthe O-helix region, of the Thermotoga neapolitana polymerase gene.

FIG. 6A schematically depicts the construction of plasmids pUC-Tne(3′→5′) and pUC-Tne FY.

FIG. 6B schematically depicts the construction of plasmids pTrc Tne35and pTrcTne FY.

FIG. 7 schematically depicts the construction of plasmid pTrcTne35 FY.

FIG. 8 schematically depicts the construction of plasmid pTTQTne5 FY andpTTQTne535FY.

FIG. 9 depicts a gel containing two sequencing reaction sets showing theefficient ³⁵S incorporation by Tne DNA polymerase of Example 12.Alkali-denatured pUC19 DNA was sequenced with Tne DNA polymerase in setA. M13 mp19(+) DNA was sequenced in set B.

FIG. 10 depicts a gel containing three sequencing reaction sets showingthat the mutant Tne DNA polymerase of Example 12 generates clearsequence from plasmids containing cDNAs with poly(dA) tails.Alkali-denatured plasmid DNAs containing cDNA inserts were sequencedusing either Tne DNA polymerase (sets A and B), or Sequenase Ver 2.0(set C).

FIG. 11 depicts a gel containing three sequencing reaction sets thatcompare the mutant Tne DNA polymerase of Example 12 (set A), Sequenase™(set B) and Taq DNA polymerase (set C) generated sequences from aplasmid containing poly(dC).

FIG. 12 depicts a gel containing three sequencing reaction sets showingthat the mutant Tne DNA polymerase of Example 12 (set A) produces³⁵-labeled sequence 3-fold stronger than Thermo Sequenase™ (set B) andwithout the uneven band intensities obtained with Taq DNA polymerase(set C).

FIG. 13 depicts a gel containing four sequencing reaction setsdemonstrating that the mutant Tne DNA polymerase of Example 12 produceshigh quality sequences of in vitro amplified DNA (set A, E. coli βpolI(˜450 bp); set B, E. coli rrsE (˜350 bp); set C, ori from pSC101 (˜1.5kb); and set D, an exon from human HSINF gene (˜750 bp).

FIGS. 14A and 14B depict gels containing three and four sequencingreaction sets, respectively, showing that the mutant Tne DNA polymeraseof Example 12 provides superior sequence from double-stranded DNA clonescontaining poly(dA) or poly(dC) stretches. FIG. 14A, supercoiled plasmidDNAs containing inserts with homopolymers were cycle sequenced using themutant Tne DNA polymerase (set A, RPA1; set B, elf (cap bindingprotein); and set C, a poly(dC)-tailed 5′ RACE-derived insert). FIG.14B, supercoiled plasmid DNAs containing inserts with homopolymers werecycled sequenced using Taq DNA polymerase (set D), or SequiTherm™ (setsE-G) (set D, RPA; set E, RPA; set F, a poly(dC)-tailed 5′ RACE-derivedinsert; and set G, elf).

FIG. 15 depicts a gel containing two sequencing reaction sets showingcycle sequencing using the mutant Tne DNA polymerase of Example 12 and³²P end-labeled primer.

FIGS. 16A-16C and 16D-16F depict two sets of chromatograms showingcomparison of the mutant Tne DNA polymerase of Example 12 (16A-16C) toAmpliTaq FS™ (16D-16F) in Fluorescent Dye Primer Sequencing.

FIGS. 17A-17C and 17D-17F depict chromatograms showing a comparison ofthe mutant Tne DNA polymerase of Example 12 (17A) to AmpliTaq FS™ (17B)in Fluorescent Dye Terminator Sequencing.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

In the description that follows, a number of terms used in recombinantDNA technology are extensively utilized. In order to provide a clearerand consistent understanding of the specification and claims, includingthe scope to be given such terms, the following definitions areprovided.

Cloning vector. A plasmid, cosmid or phage DNA or other DNA moleculewhich is able to replicate autonomously in a host cell, and which ischaracterized by one or a small number of restriction endonucleaserecognition sites at which such DNA sequences may be cut in adeterminable fashion without loss of an essential biological function ofthe vector, and into which DNA may be spliced in order to bring aboutits replication and cloning. The cloning vector may further contain amarker suitable for use in the identification of cells transformed withthe cloning vector. Markers, for example, are tetracycline resistance orampicillin resistance.

Expression vector. A vector similar to a cloning vector but which iscapable of enhancing the expression of a gene which has been cloned intoit, after transformation into a host. The cloned gene is usually placedunder the control of (i.e., operably linked to) certain controlsequences such as promoter sequences.

Recombinant host. Any prokaryotic or eukaryotic or microorganism whichcontains the desired cloned genes in an expression vector, cloningvector or any DNA molecule. The term “recombinant host” is also meant toinclude those host cells which have been genetically engineered tocontain the desired gene on the host chromosome or genome.

Host. Any prokaryotic or eukaryotic microorganism that is the recipientof a replicable expression vector, cloning vector or any DNA molecule.The DNA molecule may contain, but is not limited to, a structural gene,a promoter and/or an origin of replication.

Promoter. A DNA sequence generally described as the 5′ region of a gene,located proximal to the start codon. At the promoter region,transcription of an adjacent gene(s) is initiated.

Gene. A DNA sequence that contains information necessary for expressionof a polypeptide or protein. It includes the promoter and the structuralgene as well as other sequences involved in expression of the protein.

Structural gene. A DNA sequence that is transcribed into messenger RNAthat is then translated into a sequence of amino acids characteristic ofa specific polypeptide.

Operably linked. As used herein means that the promoter is positioned tocontrol the initiation of expression of the polypeptide encoded by thestructural gene.

Expression. Expression is the process by which a gene produces apolypeptide. It includes transcription of the gene into messenger RNA(mRNA) and the translation of such mRNA into polypeptide(s).

Substantially Pure. As used herein “substantially pure” means that thedesired purified protein is essentially free from contaminating cellularcontaminants which are associated with the desired protein in nature.Contaminating cellular components may include, but are not limited to,phosphatases, exonucleases, endonucleases or undesirable DNA polymeraseenzymes.

Primer. As used herein “primer” refers to a single-strandedoligonucleotide that is extended by covalent bonding of nucleotidemonomers during amplification or polymerization of a DNA molecule.

Template. The term “template” as used herein refers to a double-strandedor single-stranded DNA molecule which is to be amplified, synthesized orsequenced. In the case of a double-stranded DNA molecule, denaturationof its strands to form a first and a second strand is performed beforethese molecules may be amplified, synthesized or sequenced. A primer,complementary to a portion of a DNA template is hybridized underappropriate conditions and the DNA polymerase of the invention may thensynthesize a DNA molecule complementary to said template or a portionthereof. The newly synthesized DNA molecule, according to the invention,may be equal or shorter in length than the original DNA template.Mismatch incorporation during the synthesis or extension of the newlysynthesized DNA molecule may result in one or a number of mismatchedbase pairs. Thus, the synthesized DNA molecule need not be exactlycomplementary to the DNA template.

Incorporating. The term “incorporating” as used herein means becoming apart of a DNA molecule or primer.

Amplification. As used herein “amplification” refers to any in vitromethod for increasing the number of copies of a nucleotide sequence withthe use of a DNA polymerase. Nucleic acid amplification results in theincorporation of nucleotides into a DNA molecule or primer therebyforming a new DNA molecule complementary to a DNA template. The formedDNA molecule and its template can be used as templates to synthesizeadditional DNA molecules. As used herein, one amplification reaction mayconsist of many rounds of DNA replication. DNA amplification reactionsinclude, for example, polymerase chain reactions (PCR). One PCR reactionmay consist of 30 to 100 “cycles” of denaturation and synthesis of a DNAmolecule.

Oligonucleotide. “Oligonucleotide” refers to a synthetic or naturalmolecule comprising a covalently linked sequence of nucleotides whichare joined by a phosphodiester bond between the 3′ position of thepentose of one nucleotide and the 5′ position of the pentose of theadjacent nucleotide.

Nucleotide. As used herein “nucleotide” refers to a base-sugar-phosphatecombination. Nucleotides are monomeric units of a nucleic acid sequence(DNA and RNA). The term nucleotide includes deoxyribonucleosidetriphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivativesthereof. Such derivatives include, for example, [αS]dATP, 7-deaza-dGTPand 7-deaza-dATP. The term nucleotide as used herein also refers todideoxyribonucleoside triphosphates (ddNTPs) and their derivatives.Illustrated examples of dideoxyribonucleoside triphosphates include, butare not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. According tothe present invention, a “nucleotide” may be unlabeled or detectablylabeled by well known techniques. Detectable labels include, forexample, radioactive isotopes, fluorescent labels, chemiluminescentlabels, bioluminescent labels and enzyme labels.

Thermostable. As used herein “thermostable” refers to a DNA polymerasewhich is resistant to inactivation by heat. DNA polymerases synthesizethe formation of a DNA molecule complementary to a single-stranded DNAtemplate by extending a primer in the 5′-to-3′ direction. This activityfor mesophilic DNA polymerases may be inactivated by heat treatment. Forexample, T5 DNA polymerase activity is totally inactivated by exposingthe enzyme to a temperature of 90° C. for 30 seconds. As used herein, athermostable DNA polymerase activity is more resistant to heatinactivation than a mesophilic DNA polymerase. However, a thermostableDNA polymerase does not mean to refer to an enzyme which is totallyresistant to heat inactivation and thus heat treatment may reduce theDNA polymerase activity to some extent. A thermostable DNA polymerasetypically will also have a higher optimum temperature than mesophilicDNA polymerases.

Hybridization. The terms “hybridization” and “hybridizing” refers to thepairing of two complementary single-stranded nucleic acid molecules (RNAand/or DNA) to give a double-stranded molecule. As used herein, twonucleic acid molecules may be hybridized, although the base pairing isnot completely complementary. Accordingly, mismatched bases do notprevent hybridization of two nucleic acid molecules provided thatappropriate conditions, well known in the art, are used.

3′-to-5′ Exonuclease Activity. “3′-to-5′ exonuclease activity” is anenzymatic activity well known to the art. This activity is oftenassociated with DNA polymerases, and is thought to be involved in a DNAreplication “editing” or correction mechanism.

A “DNA polymerase substantially reduced in 3′-to-5′ exonucleaseactivity” is defined herein as either (1) a mutated DNA polymerase thathas about or less than 10%, or preferably about or less than 1%, of the3′-to-5′ exonuclease activity of the corresponding unmutated, wild-typeenzyme, or (2) a DNA polymerase having a 3′-to-5′ exonuclease specificactivity which is less than about 1 unit/mg protein, or preferably aboutor less than 0.1 units/mg protein. A unit of activity of 3′-to-5′exonuclease is defined as the amount of activity that solubilizes 10nmoles of substrate ends in 60 min. at 37° C., assayed as described inthe “BRL 1989 Catalogue & Reference Guide”, page 5, with HhaI fragmentsof lambda DNA 3′-end labeled with [³H]dTTP by terminal deoxynucleotidyltransferase (TdT). Protein is measured by the method of Bradford, Anal.Biochem. 72:248 (1976). As a means of comparison, natural, wild-typeT5-DNA polymerase (DNAP) or T5-DNAP encoded by pTTQ19-T5-2 has aspecific activity of about 10 units/mg protein while the DNA polymeraseencoded by pTTQ19-T5-2(Exo⁻) (U.S. Pat. No. 5,270,179) has a specificactivity of about 0.0001 units/mg protein, or 0.001% of the specificactivity of the unmodified enzyme, a 10⁵-fold reduction.

5′-to-3′ Exonuclease Activity. “5′-to-3′ exonuclease activity” is alsoan enzymatic activity well known in the art. This activity is oftenassociated with DNA polymerases, such as E. coli PolI and PolIII.

A “DNA polymerase substantially reduced in 5′-to-3′ exonucleaseactivity” is defined herein as either (1) a mutated DNA polymerase thathas about or less than 10%, or preferably about or less than 1%, of the5′-to-3′ exonuclease activity of the corresponding unmutated, wild-typeenzyme, or (2) a DNA polymerase having 5′-to-3′ exonuclease specificactivity which is less than about 1 unit mg protein, or preferably aboutor less than 0.1 units/mg protein.

Both of the 3′-to-5′ and 5′-to-3′ exonuclease activities can be observedon sequencing gels. Active 5′-to-3′ exonuclease activity will producenonspecific ladders in a sequencing gel by removing nucleotides from the5′-end of the growing primers. 3′-to-5′ exonuclease activity can bemeasured by following the degradation of radiolabeled primers in asequencing gel. Thus, the relative amounts of these activities, e.g. bycomparing wild-type and mutant polymerases, can be determined with nomore than routine experimentation.

1. Cloning and Expression of Thermotoga DNA Polymerases

The Thermotoga DNA polymerase of the invention can be isolated from anystrain of Thermotoga which produces a DNA polymerase. The preferredstrain to isolate the gene encoding Thermotoga DNA polymerase of thepresent invention is Thermotoga neapolitana (Tne) and Thermotogamaritima (Tma). The most preferred Thermotoga neapolitana for isolatingthe DNA polymerase of the invention was isolated from an Africancontinental solfataric spring (Windberger et al., Arch. Microbiol.151:506-512 (1989) and may be obtained from Deutsche Sammalung vonMicroorganismen und Zellkulturan GmbH (DSM; German Collection ofMicroorganisms and Cell Culture) Mascheroder Weg lb D-3300 Braunschweig,Federal Republic of Germany, as Deposit No. 5068 (deposited Dec. 13,1988).

To clone a gene encoding a Thermotoga DNA polymerase of the invention,isolated DNA which contains the polymerase gene obtained from Thermotogacells, is used to construct a recombinant DNA library in a vector. Anyvector, well known in the art, can be used to clone the wild type ormutant Thermotoga DNA polymerase of the present invention. However, thevector used must be compatible with the host in which the recombinantDNA library will be transformed.

Prokaryotic vectors for constructing the plasmid library includeplasmids such as those capable of replication in E. coli such as, forexample, pBR322, ColE1, pSC101, pUC-vectors (pUC18, pUC19, etc.: In:Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1982); and Sambrook et al., In:Molecular Cloning A Laboratory Manual (2d ed.) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). Bacillus plasmidsinclude pC194, pC221, pC217, etc. Such plasmids are disclosed byGlyczan, T. In: The Molecular Biology Bacilli, Academic Press, York(1982), 307-329. Suitable Streptomyces plasmids include pIJ101 (Kendallet al., J. Bacteriol 169:4177-4183 (1987)). Pseudomonas plasmids arereviewed by John et al., (Rad. Insec. Dis. 8:693-704 (1986)), and Igaki,(Jpn. J. Bacteriol. 33:729-742 (1978)). Broad-host range plasmids orcosmids, such as pCP13 (Darzins and Chakrabarbary, J. Bacteriol.159:9-18, 1984) can also be used for the present invention. Thepreferred vectors for cloning the genes of the present invention areprokaryotic vectors. Preferably, pCP13 and pUC vectors are used to clonethe genes of the present invention.

The preferred host for cloning the wild type or mutant DNA polymerasegenes of the invention is a prokaryotic host. The most preferredprokaryotic host is E. coli. However, the wild type or mutant DNApolymerase genes of the present invention may be cloned in otherprokaryotic hosts including, but not limited to, Escherichia, Bacillus,Streptomyces, Pseudomonas, Salmonella, Serratia, and Proteus. Bacterialhosts of particular interest include E. coli DH10B, which may beobtained from Life Technologies, Inc. (LTI) (Gaithersburg, Md.).

Eukaryotic hosts for cloning and expression of the wild type or mutantDNA polymerases of the present invention include yeast, fungi, andmammalian cells. Expression of the desired DNA polymerase in sucheukaryotic cells may require the use of eukaryotic regulatory regionswhich include eukaryotic promoters. Cloning and expressing the wild typeor mutant DNA polymerase gene of the invention in eukaryotic cells maybe accomplished by well known techniques using well known eukaryoticvector systems.

Once a DNA library has been constructed in a particular vector, anappropriate host is transformed by well known techniques. Transformedcolonies are plated at a density of approximately 200-300 colonies perpetri dish. Colonies are then screened for the expression of a heatstable DNA polymerase by transferring transformed E. coli colonies tonitrocellulose membranes. After the transferred cells are grown onnitrocellulose (approximately 12 hours), the cells are lysed by standardtechniques, and the membranes are then treated at 95° C. for 5 minutesto inactivate the endogenous E. coli enzyme. Other temperatures may beused to inactivate the host polymerases depending on the host used andthe temperature stability of the DNA polymerase to be cloned. Stable DNApolymerase activity is then detected by assaying for the presence of DNApolymerase activity using well known techniques. Sagner et al., Gene97:119-123 (1991), which is hereby incorporated by reference in itsentirety. The gene encoding a DNA polymerase of the present inventioncan be cloned using the procedure described by Sagner et al., supra.

The recombinant host containing the wild type gene encoding Tne DNApolymerase, E. coli DH10B (pUC-Tne), was deposited on Sep. 30, 1994,with the Collection, Agricultural Research Culture Collection (NRRL),1815 North University Street, Peoria, Ill. 61604 USA as Deposit No. NRRLB-21338. The gene encoding Tma DNA polymerase has also been cloned andsequenced (U.S. Pat. No. 5,374,553, which is expressly incorporated byreference in its entirety).

If the Thermotoga (e.g., Tne or Tma) DNA polymerase has 3′-to-5′exonuclease activity, this activity may be reduced, substantiallyreduced, or eliminated by mutating the DNA polymerase gene. Suchmutations include point mutations, frame shift mutations, deletions andinsertions. Preferably, the region of the gene encoding the 3′-to-5′exonuclease activity is mutated or deleted using techniques well knownin the art (Sambrook et al., (1989) in: Molecular Cloning, A LaboratoryManual (2nd Ed.), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

The 3′-to-5′ exonuclease activity can be reduced or impaired by creatingsite specific mutants within the 3′→5′ exonuclease domain. See infra. Ina specific embodiment of the invention AsP³²³ of Tne DNA polymerase (SEQID NO. 3) is changed to any amino acid, preferably to Ala³²³ tosubstantially reduce 3′-to-5′ exonuclease activity. In another specificembodiment of the invention, AsP³²³ of Tma may be changed to any otheramino acid, preferably to Ala to substantially reduce 3′-to-5′exonuclease activity.

The 5′→3′ exonuclease activity of the DNA polymerase can be reduced oreliminated by mutating the DNA polymerase gene. Such mutations includepoint mutations, frame shift mutations, deletions, and insertions.Preferably, the region of the gene encoding the 5′→3′ exonucleaseactivity is deleted using techniques well known in the art. Inembodiments of this invention, any one of six conserved amino acids thatare associated with the 5′→3′ exonuclease activity can be mutated.Examples of these conserved amino acids with respect to Tne DNApolymerase include Asp⁸, Glu¹¹², Asp¹¹⁴, Asp¹¹⁵, Asp¹³⁷, and Asp¹³⁹.Other possible sites for mutation are: Gly¹⁰², Gly¹⁸⁷ and Gly¹⁹⁵.

The present invention is directed broadly to mutations of DNApolymerases that result in the reduction or elimination of 5′-3′exonuclease activity. Other particular mutations correspond to thefollowing amino acids.

E. coli polI: Asp¹³, Glu¹¹³, Asp¹¹⁵, Asp¹¹⁶, Asp¹³⁸, and Asp¹⁴⁰.

Taq pol: Asp⁸, Glu¹¹⁷, Asp¹¹⁹, Asp¹²⁰, Asp¹⁴², and Asp¹⁴⁴. Tma pol:Asp⁸, Glu¹¹², Asp¹¹⁴, Asp¹¹⁵, Asp¹³⁷, and Asp¹³⁹.

Amino acid residues of Taq DNA polymerase are as numbered in U.S. Pat.No. 5,079,352.

Amino acid residues of Thermotoga maritima (Tma) DNA polymerase arenumbered as in U.S. Pat. No. 5,374,553.

By comparison to the amino acid sequence of other DNA polymerases, thecorresponding sites can easily be located and the DNA mutanigized toprepare a coding sequence for the corresponding DNA polymerase whichlacks the 5′-3′ exonuclease activity. Examples of other DNA polymerasesthat can be so mutated include:

Enzyme or source Mutation positions Streptococcus Asp¹⁰, Glu¹¹⁴, Asp¹¹⁶,Asp¹¹⁷, Asp¹³⁹, Asp¹⁴¹ pneumoniae Thermus flavus Asp¹⁷, Glu¹¹⁶, Asp¹¹⁸,Asp¹¹⁹, Asp¹⁴¹, Asp¹⁴³ Thermus thermophilus Asp¹⁸, Glu¹¹⁸, Asp¹²⁰,Asp¹²¹, Asp¹⁴³, Asp¹⁴⁵ Deinococcus Asp¹⁸, Glu¹¹⁷, Asp¹¹⁹, Asp¹²⁰,Asp¹⁴², Asp¹⁴⁴ radiodurans Bacillus caldotenax Asp⁹, Glu¹⁰⁹, Asp¹¹¹,Asp¹¹², Asp¹³⁴, Asp¹³⁶

Coordinates of S. pneumoniae, T. flavus, D. radiodurans, B. caldotenaxwere obtained from Gutman and Minton. Coordinates of T. thermophiluswere obtained from International Patent No. WO 92/06200.

To abolish the 5′-3′ exonuclease activity, amino acids are selectedwhich have different properties. For example, an acidic amino acid suchas Asp may be changed to a basic, neutral or polar but uncharged aminoacid such as Lys, Arg, His (basic); Ala, Val, Leu, Ile, Pro, Met, Phe,Trp (neutral); or Gly, Ser, Thr, Cys, Tyr, Asn or Gln (polar butuncharged). Glu may be changed to Asp, Ala, Val Leu, Ile, Pro, Met, Phe,Trp, Gly, Ser, Thr, Cys, Tyr, Asn or Gln. Specifically, the Alasubstitution in the corresponding position is expected to abolish 5′-exoactivity.

Preferably, oligonucleotide directed mutagenesis is used to create themutant DNA polymerase which allows for all possible classes of base pairchanges at any determined site along the encoding DNA molecule. Ingeneral, this technique involves annealing a oligonucleotidecomplementary (except for one or more mismatches) to a single strandednucleotide sequence coding for the DNA polymerase of interest. Themismatched oligonucleotide is then extended by DNA polymerase,generating a double stranded DNA molecule which contains the desiredchange in sequence on one strand. The changes in sequence can of courseresult in the deletion, substitution, or insertion of an amino acid. Thedouble stranded polynucleotide can then be inserted into an appropriateexpression vector, and a mutant polypeptide can thus be produced. Theabove-described oligonucleotide directed mutagenesis can of course becarried out via PCR.

In other embodiments, the entire 5′→3′ exonuclease domain of the DNApolymerase can be deleted by proteolytic cleavage or by geneticengineering. For example, a unique SphI restriction site can be used toobtain a clone devoid of nucleotides encoding the 219 amino terminalamino acids of Tne DNA polymerase. Examples of such a clone arepTTQTne535FY and pTTQTne5FY. Alternatively, less than the 219 aminoterminal amino acids may be removed, for example, by treating the DNAcoding for the Tne DNA polymerase with an exonuclease, isolating thefragments, ligating the fragments into a cloning vehicle, transfectingcells with the cloning vehicle, and screening the transformants for DNApolymerase activity and lack of 5′→3′ exonuclease activity, with no morethan routine experimentation.

Thermotoga DNA polymerase mutants can also be made to render thepolymerase non-discriminating against non-natural nucleotides such asdideoxynucleotides. Changes within the O-helix of Thermotogapolymerases, such as other point mutations, deletions, and insertions,can be made to render the polymerase non-discriminating. By way ofexample, one Tne DNA polymerase mutant having this property substitutesa nonnatural amino acid such as Tyr for Phe at amino acid 67 as numberedin FIGS. 5A and 5B, and 730 of SEQ ID NO:3.

The O-helix region is a 14 amino acid sequence corresponding to aminoacids 722-735 of SEQ ID NO:3 or amino acids 59-72 as numbered in FIGS.5A and 5B. The O-helix may be defined as RXXXKXXXFXXXYX, wherein X isany amino acid. The most important amino acids in conferringdiscriminatory activity include Arg, Lys and Phe. Amino acids which maybe substituted for Arg at positions 722 are selected independently fromAsp, Glu, Ala, Val Leu, Ile, Pro, Met, Phe, Trp, Gly, Ser, Thr, Cys,Tyr, Gln, Asn, Lys and His. Amino acids that may be substituted for Pheat position 730 include Lys, Arg, His, Asp, Glu, Ala, Val, Leu, Ile,Pro, Met, Trp, Gly, Ser, Thr, Cys, Tyr, Asn or Gln. Amino acids that maybe substituted for Lys at position 726 of SEQ ID NO: 3 include Tyr, Arg,His, Asp, Glu, Ala, Val, Leu, Ile, Pro, Met, Trp, Gly, Ser, Thr, Cys,Phe, Asn or Gln. Preferred mutants include Tyr⁷³⁰, Ala⁷³⁰, Ser⁷³⁰ andThr¹³⁰. Such Tne mutants may be prepared by well known methods of sitedirected mutagenesis as described herein. See also Example 10.

The corresponding mutants can also be prepared from Tma DNA polymerase,including Arg⁷²², Lys⁷²⁶ and Phe⁷³⁰. Most preferred mutants includePhe⁷³⁰ to Tyr⁷³⁰, Ser⁷³⁰, Thr⁷³⁰ and Ala⁷³⁰.

2. Enhancing Expression of Thermotoga DNA Polymerase

To optimize expression of the wild type or mutant Thermotoga DNApolymerases of the present invention, inducible or constitutivepromoters are well known and may be used to express high levels of apolymerase structural gene in a recombinant host. Similarly, high copynumber vectors, well known in the art, may be used to achieve highlevels of expression. Vectors having an inducible high copy number mayalso be useful to enhance expression of Thermotoga DNA polymerase in arecombinant host.

To express the desired structural gene in a prokaryotic cell (such as,E. coli, B. subtilis, Pseudomonas, etc.), it is necessary to operablylink the desired structural gene to a functional prokaryotic promoter.However, the natural Thermotoga promoter may function in prokaryotichosts allowing expression of the polymerase gene. Thus, the naturalThermotoga promoter or other promoters may be used to express the DNApolymerase gene. Such other promoters may be used to enhance expressionand may either be constitutive or regulatable (i.e., inducible orderepressible) promoters. Examples of constitutive promoters include theint promoter of bacteriophage λ, and the bla promoter of the β-lactamasegene of pBR322. Examples of inducible prokaryotic promoters include themajor right and left promoters of bacteriophage λ (P_(R) and P_(L)),trp, recA, lacZ, lacI, tet, gal, trc, and tac promoters of E. coli. TheB. subtilis promoters include α-amylase (Ulmanen et al., J. Bacteriol162:176-182 (1985)) and Bacillus bacteriophage promoters (Gryczan, T.,In: The Molecular Biology Of Bacilli, Academic Press, New York (1982)).Streptomyces promoters are described by Ward et al., Mol. Gen. Genet.203:468-478 (1986)). Prokaryotic promoters are also reviewed by Glick,J. Ind. Microbiol. 1:277-282 (1987); Cenatiempto, Y., Biochimie68:505-516 (1986); and Gottesman, Ann. Rev. Genet. 18:415-442 (1984).Expression in a prokaryotic cell also requires the presence of aribosomal binding site upstream of the gene-encoding sequence. Suchribosomal binding sites are disclosed, for example, by Gold et al., Ann.Rev. Microbiol. 35:365404 (1981).

To enhance the expression of Thermotoga (e.g., The and Tma) DNApolymerase in a eukaryotic cell, well known eukaryotic promoters andhosts may be used. Preferably, however, enhanced expression ofThermotoga DNA polymerase is accomplished in a prokaryotic host. Thepreferred prokaryotic host for overexpressing this enzyme is E. coli.

3. Isolation and Purification of Thermotoga DNA Polymerase

The enzyme(s) of the present invention (Thermotoga DNA polymerases andmutants thereof) is preferably produced by fermentation of therecombinant host containing and expressing the cloned DNA polymerasegene. However, the wild type and mutant DNA polymerases of the presentinvention may be isolated from any Thermotoga strain which produces thepolymerase of the present invention. Fragments of the polymerase arealso included in the present invention. Such fragments includeproteolytic fragments and fragments having polymerase activity.

Any nutrient that can be assimilated by Thermotoga or a host containingthe cloned Thermotoga DNA polymerase gene may be added to the culturemedium. Optimal culture conditions should be selected case by caseaccording to the strain used and the composition of the culture medium.Antibiotics may also be added to the growth media to insure maintenanceof vector DNA containing the desired gene to be expressed. Cultureconditions for Thermotoga neapolitana have, for example, been describedby Huber et al., Arch. Microbiol. 144:324-333 (1986). Media formulationsare also described in DSM or ATCC Catalogs and Sambrook et al., In:Molecular Cloning, a Laboratory Manual (2nd ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). Thermotoga andrecombinant host cells producing the DNA polymerase of this inventioncan be separated from liquid culture, for example, by centrifugation. Ingeneral, the collected microbial cells are dispersed in a suitablebuffer, and then broken down by ultrasonic treatment or by other wellknown procedures to allow extraction of the enzymes by the buffersolution. After removal of cell debris by ultracentrifugation orcentrifugation, the DNA polymerase can be purified by standard proteinpurification techniques such as extraction, precipitation,chromatography, affinity chromatography, electrophoresis or the like.Assays to detect the presence of the DNA polymerase during purificationare well known in the art and can be used during conventionalbiochemical purification methods to determine the presence of theseenzymes.

4. Uses of Thermotoga DNA Polymerase

The wild type and mutant Thermotoga DNA polymerases (e.g., Tma and Tne)of the present invention may be used in well known DNA sequencing, DNAlabeling, DNA amplification and cDNA synthesis reactions. Thermotoga DNApolymerase mutants devoid of or substantially reduced in 3′→5′exonuclease activity, devoid of or substantially reduced in 5′→3′exonuclease activity, or containing one or mutations in the O-helix thatmake the enzyme nondiscriminatory for dNTPs and ddNTPs (e.g., aPhe⁷³⁰→Tyr⁷³⁰ mutation of SEQ ID NO: 3) are especially useful for DNAsequencing, DNA labeling, and DNA amplification reactions and cDNAsynthesis. Moreover, Thermotoga DNA polymerase mutants containing two ormore of these properties are also especially useful for DNA sequencing,DNA labeling, DNA amplification or cDNA synthesis reactions. As is wellknown, sequencing reactions (isothermal DNA sequencing and cyclesequencing of DNA) require the use of DNA polymerases. Dideoxy-mediatedsequencing involves the use of a chain-termination technique which usesa specific polymer for extension by DNA polymerase, a base-specificchain terminator and the use of polyacrylamide gels to separate thenewly synthesized chain-terminated DNA molecules by size so that atleast a part of the nucleotide sequence of the original DNA molecule canbe determined. Specifically, a DNA molecule is sequenced by using fourseparate DNA sequence reactions, each of which contains differentbase-specific terminators. For example, the first reaction will containa G-specific terminator, the second reaction will contain a T-specificterminator, the third reaction will contain an A-specific terminator,and a fourth reaction may contain a C-specific terminator. Preferredterminator nucleotides include dideoxyribonucleoside triphosphates(ddNTPs) such as ddATP, ddTTP, ddGTP, ddITP and ddCTP. Analogs ofdideoxyribonucleoside triphosphates may also be used and are well knownin the art.

When sequencing a DNA molecule, ddNTPs lack a hydroxyl residue at the 3′position of the deoxyribose base and thus, although they can beincorporated by DNA polymerases into the growing DNA chain, the absenceof the 3′-hydroxy residue prevents formation of the next phosphodiesterbond resulting in termination of extension of the DNA molecule. Thus,when a small amount of one ddNTP is included in a sequencing reactionmixture, there is competition between extension of the chain andbase-specific termination resulting in a population of synthesized DNAmolecules which are shorter in length than the DNA template to besequenced. By using four different ddNTPs in four separate enzymaticreactions, populations of the synthesized DNA molecules can be separatedby size so that at least a part of the nucleotide sequence of theoriginal DNA molecule can be determined. DNA sequencing bydideoxy-nucleotides is well known and is described by Sambrook et al.,In: Molecular Cloning, a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). As will be readilyrecognized, the Thermotoga DNA polymerases and mutants thereof of thepresent invention may be used in such sequencing reactions.

As is well known, detectably labeled nucleotides are typically includedin sequencing reactions. Any number of labeled nucleotides can be usedin sequencing (or labeling) reactions, including, but not limited to,radioactive isotopes, fluorescent labels, chemiluminescent labels,bioluminescent labels, and enzyme labels. It has been discovered thatthe wild type and mutant DNA polymerase of the present invention may beuseful for incorporating αS nucleotides ([αS]dATP, [αS]dTTP, [αS]dCTPand [αS]dGTP) during sequencing (or labeling) reactions. For example,[α³⁵S]dATP, a commonly used detectably labeled nucleotide in sequencingreactions, is incorporated three times more efficiently with the Tne DNApolymerase of the present invention, than with Taq DNA polymerase. Thus,the enzyme of the present invention is particularly suited forsequencing or labeling DNA molecules with [α³⁵S]dNTPs.

Polymerase chain reaction (PCR), a well known DNA amplificationtechnique, is a process by which DNA polymerase and deoxyribonucleosidetriphosphates are used to amplify a target DNA template. In such PCRreactions, two primers, one complementary to the 3′ termini (or near the3′-termini) of the first strand of the DNA molecule to be amplified, anda second primer complementary to the 3′ termini (or near the 3′-termini)of the second strand of the DNA molecule to be amplified, are hybridizedto their respective DNA strands. After hybridization, DNA polymerase, inthe presence of deoxyribonucleoside triphosphates, allows the synthesisof a third DNA molecule complementary to the first strand and a fourthDNA molecule complementary to the second strand of the DNA molecule tobe amplified. This synthesis results in two double stranded DNAmolecules. Such double stranded DNA molecules may then be used as DNAtemplates for synthesis of additional DNA molecules by providing a DNApolymerase, primers, and deoxyribonucleoside triphosphates. As is wellknown, the additional synthesis is carried out by “cycling” the originalreaction (with excess primers and deoxyribonucleoside triphosphates)allowing multiple denaturing and synthesis steps. Typically, denaturingof double stranded DNA molecules to form single stranded DNA templatesis accomplished by high temperatures. The wild type and mutantThermotoga DNA polymerases of the present invention are heat stable DNApolymerases, and thus will survive such thermal cycling during DNAamplification reactions. Thus, the wild type and mutant DNA polymerasesof the invention are ideally suited for PCR reactions, particularlywhere high temperatures are used to denature the DNA molecules duringamplification.

The Thermotoga DNA polymerase and mutants of the present invention (e.g.Tne and Tma) may also be used to prepare cDNA from mRNA templates. See,U.S. Pat. Nos. 5,405,776 and 5,244,797, the disclosures of which areexplicitly incorporated by reference herein. Thus, the invention alsorelates to a method of preparing cDNA from mRNA, comprising

(a) contacting mRNA with an oligo(dT) primer or other complementaryprimer to form a hybrid, and

(b) contacting said hybrid formed in step (a) with the Thermotoga DNApolymerase or mutant of the invention and the four dNTPs, whereby acDNA-RNA hybrid is obtained.

If the reaction mixture is step (b) further comprises an appropriateoligonucleotide which is complementary to the cDNA being produced, it isalso possible to obtain dsDNA following first strand synthesis. Thus,the invention is also directed to a method of preparing dsDNA with theThermotoga DNA polymerases and mutants thereof of the present invention.

5. Kits

The wild type and mutant Thermotoga DNA polymerases of the invention aresuited for the preparation of a kit. Kits comprising the wild type ormutant DNA polymerase(s) may be used for detectably labeling DNAmolecules, DNA sequencing, amplifying DNA molecules or cDNA synthesis bywell known techniques, depending on the content of the kit. See U.S.Pat. Nos. 4,962,020, 5,173,411, 4,795,699, 5,498,523, 5,405,776 and5,244,797. Such kits may comprise a carrying means beingcompartmentalized to receive in close confinement one or more containermeans such as vials, test tubes and the like. Each of such containermeans comprises components or a mixture of components needed to performDNA sequencing, DNA labeling, DNA amplification, or cDNA synthesis.

A kit for sequencing DNA may comprise a number of container means. Afirst container means may, for example, comprise a substantiallypurified sample of Thermotoga DNA polymerases or mutants thereof. Asecond container means may comprise one or a number of types ofnucleotides needed to synthesize a DNA molecule complementary to DNAtemplate. A third container means may comprise one or a number ofdifferent types of dideoxynucleoside triphosphates. A fourth containermeans may comprise pyrophosphatase. In addition to the above containermeans, additional container means may be included in the kit whichcomprise one or a number of DNA primers.

A kit used for amplifying DNA will comprise, for example, a firstcontainer means comprising a substantially pure mutant or wild typeThermotoga DNA polymerase of the invention and one or a number ofadditional container means which comprise a single type of nucleotide ormixtures of nucleotides. Various primers may or may not be included in akit for amplifying DNA.

Kits for cDNA synthesis will comprise a first container means containingthe wild type or mutant Tne DNA polymerase of the invention, a secondcontainer means will contain the four dNTPs and the third containermeans will contain oligo(dT) primer. See U.S. Pat. Nos. 5,405,776 and5,244,797. Since the Thermotoga DNA polymerases of the present inventionare also capable of preparing dsDNA, a fourth container means maycontain an appropriate primer complementary to the first strand cDNA.

Of course, it is also possible to combine one or more of these reagentsin a single tube. A detailed description of such formulations at workingconcentrations is described in the patent application entitled “StableCompositions for Nucleic Acid Amplification and Sequencing” filed onAug. 14, 1996, which is expressly incorporated by reference herein inits entirety.

When desired, the kit of the present invention may also includecontainer means which comprise detectably labeled nucleotides which maybe used during the synthesis or sequencing of a DNA molecule. One of anumber of labels may be used to detect such nucleotides Illustrativelabels include, but are not limited to, radioactive isotopes,fluorescent labels, chemiluminescent labels, bioluminescent labels andenzyme labels.

6. Advantages of the Thermotoga DNA Polymerase

Thermotoga DNA polymerases of the invention have distinct advantages inDNA sequencing. For example, when using the Tne DNA polymerase mutantsof the invention in single-extension sequencing, they generate strong,clear ³⁵S-labeled sequence, increase sequence signal to backgroundratio, generate ≧500 bases of sequence, reduce false stops in thesequencing ladder, and permit high temperature sequencing reactions. Theefficient ³⁵S incorporation by the Tne DNA polymerase mutants of theinvention can reduce template requirement 10-fold, give sharper bandsthan ³²P, emit lower energy radiation than ³²P, and have a longer shelflife than ³²P. Further, the Tne polymerase mutants produce longersequence reads and gives more accurate sequence interpretation. Inaddition, the use of a 70° C. reaction temperature with thisthermophilic polymerase increases sequencing efficiency ofstructure-containing and GC-rich templates.

Compared to modified T7 DNA polymerase (Sequenase™), Tne DNA polymerasemutants allow improved sequencing efficiency of structure containing andGC-rich templates, are more forgiving in incubation times for labelingand extensions, and allow one to obtain full length sequence fromone-tenth the amount of template. With regard to other polymerases, theTne DNA polymerase mutants provide, under appropriate reactionconditions, more even band intensities and give longer, more accuratesequence reads, exhibit no weak or absent “dropout” bands, exhibitimproved sequencing efficiency of structure containing and GC-richtemplates, exhibit no sequence artifacts from templates containinghomopolymers, and provide for shorter film exposure and/or less templateinput due to the efficient ³⁵S-dNTP incorporation.

With regard to cycle sequencing, the Tne DNA polymerase mutants generatestrong, clear ³⁵S-labeled sequence, they increase sequence signal tobackground ratio, generate ≧500 bases of sequence, reduce false stops inthe sequencing ladder under appropriate conditions, and permit hightemperature reactions. The Tne DNA polymerase mutants also allow forhighly efficient ³⁵S dATP incorporation and therefore shorter filmexposures and/or less template input, give sharper bands than ³²P, giveoff lower energy radiation than ³²P and have a longer shelf life than³²P. The Tne DNA polymerase mutants also produce longer sequence readsand give more accurate sequence interpretation. ³²P end labeling ofprimers generates data with less background from less pure DNA andrequires as little as 5 fmole (0.01 μg) of DNA.

With regard to cycle sequencing, compared to the mutant Taq DNApolymerase (ThermoSequenase™), the Tne DNA polymerase mutants generatethree times stronger ³⁵S-labeled sequence without an extra 2 hour cycledlabeling step, require no special primer design for ³⁵S labeling, andallow for sequencing of PCR products directly using any primer. Comparedto SequiTherm™, the mutants of Tne DNA polymerase generate three timesstronger ³⁵S-labeled sequence, give more even band intensities, giveslonger and more accurate sequence reads, require less template and lessprimer, and give no sequence artifacts from templates containinghomopolymers. Compared to various other polymerases (e.g. Tth DNApolymerase), the Tne DNA polymerase mutants under appropriate reactionconditions generate three times stronger ³⁵S-labeled sequence, give moreeven band intensities, give longer and more accurate sequence reads,give no weak or absent “dropout” bands, improve sequencing efficiency ofstructure-containing and GC-rich templates, and reduce false stops insequencing ladders, including through homopolymer regions.

With regard to fluorescent sequencing, the mutants of Tne DNA polymerasereadily accept dye primers and dye terminators, increase sequence signalto background ratio, produce fewer ambiguous calls, and generate ≧500bases of sequence. The Tne DNA polymerase mutants also produce longersequence read lengths, give more accurate sequence interpretation, andallow for quantitation of bases in heterologous mixtures. Since the TneDNA polymerase mutants provide for good incorporation of dyeterminators, such dye terminators can be reduced 500-fold. Further,increased signal improves bases calling, reduces cost and time tosequence, eliminates the need to remove excess dye terminators beforegel loading, and produces more even band intensities. The efficient useof dye primers generates data with less background from impure DNA andrequires as little as 0.6 μg of dsDNA (double-stranded DNA).

With regard to the use of Thermo Sequenase™ and AmpliTaq FS™ influorescent sequencing, the Tne DNA polymerase mutants provide more evenband intensities in dye terminator sequencing and give comparableresults with dye primers. With regard to SequiTherm™, the Tne DNApolymerase mutants give more even band intensities that give longer,more accurate sequencing reads with both dye terminators and dyeprimers, use 500-fold less dye terminators, eliminate post reactionclean up of dye terminators, require 10-fold less template, and allowfor quantitation of bases in heterologous mixtures using dye primers.

With regard to the use of various other enzymes in fluorescentsequencing, such as AmpliTaq™ and AmpliTaqCS™, mutant Tne DNApolymerases under appropriate reaction conditions provide more even bandintensities and more accurate sequence reads with both dye terminatorsand dye primers, give no weak or absent “dropout” bands, have lowerbackground and fewer false stops, use 500-fold less dye terminators,eliminate post reaction clean up of dye terminators, require 10-foldless template, and allow for quantitation of bases in heterologousmixtures.

As shown in FIG. 3, Tne DNA polymerase incorporates α-thio dATP at threetimes the rate of Taq DNA polymerase. However, surprisingly, when α-thiodATP is used in place of dATP in sequencing reactions using [α-³⁵S]dATPand mutants of Tne DNA polymerase, the resulting sequencing band signalintensity is increased by approximately 8-10 fold. The weak signal seenwhen dATP is used reflects the mutant DNA polymerase's strong preferencefor incorporating dATP over α-thio dATP from a mixed pool. Attempts toimprove signal intensity by merely decreasing the amount of dATPresulted in very poor quality sequence with many false stops. Parallelexperiments with [α-³²P]dATP and low concentrations of dATP producedsimilar poor quality sequence, indicating that the nucleotideconcentration imbalance was causing the enzyme to perform poorly. Byusing α-thio dATP mixed with [α-³⁵S]dATP, the four nucleotideconcentrations kept constant without diminishing signal or sequencequality.

Having now generally described the invention, the same will be morereadily understood through reference to the following Examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

Example 1 Bacterial Strains And Growth Conditions

Thermotoga neapolitana DSM No. 5068 was grown under anaerobic conditionsas described in the DSM catalog (addition of resazurin, Na₂S, and sulfurgranules while sparging the media with nitrogen) at 85° C. in an oilbath from 12 to 24 hours. The cells were harvested by filtering thebroth through Whatman #1 filter paper. The supernatant was collected inan ice bath and then centrifuged in a refrigerated centrifuge at 8,000rpms for twenty minutes. The cell paste was stored at −70° C. prior tototal genomic DNA isolation.

E. coli strains were grown in 2×LB broth base (Lennox L broth base:GIBCO/BRL) medium. Transformed cells were incubated in SOC (2% tryptone,0.5% yeast extract, yeast 10 mM NaCl, 2.5 mM KCl, 20 mM glucose, 10 mMMgCl₂, and 10 mM MgSO₄ per liter) before plating. When appropriateantibiotic supplements were 20 mg/l tetracycline and 100 mg/lampicillin. E. coli strain DH10B (Lorow et al., Focus 12:19-20 (1990))was used as host strain. Competent DH10B may be obtained from LifeTechnologies, Inc. (LTI) (Gaithersburg, Md.).

Example 2 DNA Isolation

Thermotoga neapolitana chromosomal DNA was isolated from 1.1 g of cellsby suspending the cells in 2.5 ml TNE (50 mM Tris-HCl, pH 8.0, 50 mMNaCl, 10 mM EDTA) and treated with 1% SDS for 10 minutes at 37° C. DNAwas extracted with phenol by gently rocking the lysed cells overnight at4° C. The next day, the lysed cells were extracted withchloroform:isoamyl alcohol. The resulting chromosomal DNA was furtherpurified by centrifugation in a CsCl density gradient. Chromosomal DNAisolated from the density gradient was extracted three times withisopropanol and dialyzed overnight against a buffer containing 10 mMTris-HCl (pH 8.0) and 1 mM EDTA (TE).

Example 3 Construction of Genomic Libraries

The chromosomal DNA isolated in Example 2 was used to construct agenomic library in the plasmid pCP13. Briefly, 10 tubes each containing10 μg of Thermotoga neapolitana chromosomal DNA was digested with 0.01to 10 units of Sau3Al for 1 hour at 37° C. A portion of the digested DNAwas tested in an agarose (1.2%) gel to determine the extent ofdigestion. Samples with less than 50% digestion were pooled, ethanolprecipitated and dissolved in TE. 6.5 μg of partially digestedchromosomal DNA was ligated into 1.5 μg of pCP13 cosmid which had beendigested with BamHI restriction endonuclease and dephosphorylated withcalf intestinal alkaline phosphatase. Ligation of the partially digestedThermotoga DNA and BamHI cleaved pCP13 was carried out with T4 DNAligase at 22° C. for 16 hours. After ligation, about 1 μg of ligated DNAwas packaged using λ-packaging extract (obtained from Life Technologies,Inc., Gaithersburg, Md.). DH10B cells (Life Tech. Inc.) were theninfected with 100 μl of the packaged material. The infected cells wereplated on tetracycline containing plates. Serial dilutions were made sothat approximately 200 to 300 tetracycline resistant colonies wereobtained per plate.

Example 4 Screening for Clones Expressing Thermotoga neapolitana DNAPolymerase

Identification of the Thermotoga neapolitana DNA polymerase gene of theinvention was cloned using the method of Sagner et al., Gene 97:119-123(1991) which reference is herein incorporated in its entirety. Briefly,the E. coli tetracycline resistant colonies from Example 3 weretransferred to nitrocellulose membranes and allowed to grow for 12hours. The cells were then lysed with the fumes of chloroform:toluene(1:1) for 20 minutes and dried for 10 minutes at room temperature. Themembranes were then treated at 95° C. for 5 minutes to inactivate theendogenous E. coli enzymes. Surviving DNA polymerase activity wasdetected by submerging the membranes in 15 ml of polymerase reaction mix(50 mM Tris-HCl (pH 8.8), 1 mM MgCl₂, 3 mM β-mercaptoethanol, 10 μMdCTP, dGTP, dTTP, and 15 μCi of 3,000 Ci/mmol [α³²P]dATP) for 30 minutesat 65° C.

Using autoradiography, three colonies were identified that expressed aThermotoga neapolitana DNA polymerase. The cells were grown in liquidculture and the protein extract was made by sonication. The presence ofthe cloned thermostable polymerase was confirmed by treatment at 90° C.followed by measurement of DNA polymerase activity at 72° C. byincorporation of radioactive deoxyribonucleoside triphosphates into acidinsoluble DNA. One of the clones, expressing Tne DNA polymerase,contained a plasmid designated pCP13-32 and was used for further study.

Example 5 Subcloning of Tne DNA Polymerase

Since the pCP13-32 clone expressing the Tne DNA polymerase gene containsabout 25 kb of T. neapolitana DNA, subcloning a smaller fragment of theTne polymerase gene was attempted. The molecular weight of the Tne DNApolymerase purified from E. coli/pCP13-32 was about 100 kd. Therefore, a2.5-3.0 kb DNA fragment will be sufficient to code for full-lengthpolymerase. A second round of Sau3A partial digestion similar to Example3 was done using pCP13-32 DNA. In this case, a 3.5 kb region was cut outfrom the agarose gel, purified by Gene Clean (BIO 101, La Jolla, Calif.)and ligated into plasmid pSport 1 (Life Technologies, Inc.) which hadbeen linearized with BamHI and dephosphorylated with calf intestinalalkaline phosphatase. After ligation, DH10B was transformed and colonieswere tested for DNA polymerase activity as described in Example 4.Several clones were identified that expressed Tne DNA polymerase. One ofthe clones (pSport-Tne) containing about 3 kb insert was furthercharacterized. A restriction map of the DNA fragment is shown in FIG. 4.Further, a 2.7 Kb HindIII-SstI fragment was subcloned into pUC19 togenerate pUC19-Tne. E. coli/pUC19-Tne also produced Tne DNA polymerase.

The Tne polymerase clone was sequenced by methods known in the art. Thenucleotide sequence obtained of the 5′ end prior to the start ATG isshown in SEQ ID NO:1. The nucleotide sequence obtained which encodescarboxy-terminal region of the Tne polymerase is shown in FIGS. 5A and5B (SEQ ID NO:17). When SEQ ID NO:17 is translated it does not producethe entire amino acid sequence of the Tne polymerase due to frame shifterrors generated during the determination of the nucleotide sequence.However, an amino acid sequence of the Tne polymerase was obtained bytranslating all three reading frames of SEQ ID NO:17, comparing thesesequences with known polymerase amino acid sequences, and splicing theTne polymerase sequence together to form the amino acid sequence setforth in SEQ ID NO:18. The complete nucleotide sequence coding for Tneis shown in SEQ ID NO:2 and the complete amino acid sequence is shown inSEQ ID NO:3.

SEQ ID NO:3 shows that the Tne sequence has an N-terminal methionine. Itis not known with certainty whether the wild type Tne protein comprisesan N-terminal methionine. It is possible to remove this N-terminalmethionine according to methods well known to those of ordinary skill inthe art, e.g. with a methionine amino peptidase.

Example 6 Purification of Thermotoga neapolitana DNA Polymerase from E.coli

Twelve grams of E. coli cells expressing cloned Tne DNA polymerase(DH10B/pSport-Tne) were lysed by sonication (four thirty-second burstswith a medium tip at the setting of nine with a Heat Systems UltrasonicsInc., model 375 sonicator) in 20 ml of ice cold extraction buffer (50 mMTris HCl (pH 7.4), 8% glycerol, 5 mM mercaptoethanol, 10 mM NaCl, 1 mMEDTA, 0.5 mM PMSF). The sonicated extract was heated at 80° C. for 15min. and then cooled in ice for 5 min. 50 mM KCl and PEI (0.4%) wasadded to remove nucleic acids. The extract was centrifuged forclarification. Ammonium sulfate was added to 60%, the pellet wascollected by centrifugation and resuspended in 10 ml of column buffer(25 mM Tris-HCl (pH 7.4), 8% glycerol, 0.5% EDTA, 5 mM2-mercaptoethanol, 10 mM KCl). A Blue-Sepharose (Pharmacia) column, orpreferably a Toso heparin (Tosohaas) column, was washed with 7 columnvolumes of column buffer and eluted with a 15 column volume gradient ofbuffer from 10 mM to 2 M KCl. Fractions containing polymerase activitywere pooled. The fractions were dialyzed against 20 volumes of columnbuffer. The pooled fractions were applied to a Toso650Q column(Tosohaas). The column was washed to baseline OD₂₈₀ and elution effectedwith a linear 10 column volume gradient of 25 mM Tris (pH 7.4), 8%glycerol, 0.5 mM EDTA, 10 mM KCl, 5 mM β-mercaptoethanol to the samebuffer plus 650 mM KCl. Active fractions were pooled.

Example 7 Characterization of Purified Tne DNA Polymerase

1. Determination of the Molecular Weight of Thermotoga neapolitana DNAPolymerase

The molecular weight of 100 kilodaltons was determined byelectrophoresis in a 12.5% SDS gel by the method of Laemmli, U.K.,Nature (Lond.) 227:680-685 (1970). Proteins were detected by stainingwith Coomassie brilliant blue. A 10 kd protein ladder (LifeTechnologies, Inc.) was used as a standard.

2. Method for Measuring Incorporation of [α³⁵S]-dATP Relative to ³H-dATP

Incorporation of [αS]dATP was evaluated in a final volume of 500 μl ofreaction mix, which was preincubated at 72° C. for five minutes,containing either a [³H]TTP nucleotide cocktail (100 μM each TTP, dATP,dCTP, dGTP with [³H]TTP at 90.3 cpm/pmol), a nucleotide cocktailcontaining [αS]dATP as the only source of dATP (100 μM each [αS]dATP,dCTP, dGTP, TTP with [α³⁵S]dATP at 235 cpm/pmol), or a mixed cocktail(50 μM [αS]dATP, 50 μM dATP, 100 μM TTP, 100 μM dCTP, 100 μM dGTP with[³⁵αS] dATP at 118 cpm/pmol and [³H]TTP at 45.2 cpm/pmol) and 50 mMbicine, pH 8.5, 30 mM MgCl₂, 0.25 mg/ml activated salmon sperm DNA, 20%glycerol. The reaction was initiated by the addition of 0.3 units of T.neapolitana DNA polymerase or T. aquaticus DNA polymerase. At the timesindicated a 25 μl aliquot was removed and quenched by addition of icecold EDTA to a final concentration of 83 mM. 20 μl aliquots of thequenched reaction samples were spotted onto GF/C filters. Rates ofincorporation were compared and expressed as a ratio of T. neapolitanato T. aquaticus. The incorporation of [α³⁵S]dATP by T. neapolitana DNApolymerase was three-fold higher than that of T. aquaticus DNApolymerase.

Example 8 Reverse Transcriptase Activity

(A)_(n):(dT)₁₂₋₁₈ is the synthetic template primer used most frequentlyto assay for reverse transcriptase activity of DNA polymerases. It isnot specific for retroviral-like reverse transcriptase, however, beingcopied by many prokaryotic and eukaryotic DNA polymerases (Modak andMarcus, J. Biol. Chem. 252:11-19 (1977); Gerard et al., Biochem.13:1632-1641 (1974); Spadari and Weissbach, J. Biol. Chem. 249:5809-5815(1974)). (A)_(n):(dT)₁₂₋₁₈ is copied particularly well by cellular,replicative DNA polymerases in the presence of Mn⁺⁺, and much lessefficiently in the presence of Mg (Modak and Marcus, J. Biol. Chem.252:11-19 (1977); Gerard et al., Biochem. 13:1632-1641 (1974); Spadariand Weissbach, J. Biol. Chem. 249:5809-5815 (1974)). In contrast, mostcellular, replicative DNA polymerases do not copy the synthetic templateprimer (C)_(n):(dG)₁₂₋₁₈ efficiently in presence of either Mn⁺⁺ or Mg⁺⁺,but retroviral reverse transcriptases do. Therefore, in testing for thereverse transcriptase activity of a DNA polymerase with synthetictemplate primers, the stringency of the test increases in the followingmanner from least to most stringent: (A)_(n):(dT)₁₂₋₁₈(Mn⁺⁺)<(A)_(n):(dT)₁₂₋₁₈ (Mg⁺⁺)<<(C)_(n):(dG)₁₂₋₁₈(Mn⁺⁺)<(C)_(n):(dG)₁₂₋₁₈ (Mg⁺⁺).

The reverse transcriptase activity of Tne DNA polymerase was comparedwith Thermus thermophilus (Tth) DNA polymerase utilizing both(A)_(n):(dT)₂₀ and (C)_(n):(dG)₁₂₋₁₈. Reaction mixtures (50 μl) with(A)_(n):(dT)₂₀ contained 50 mM Tris-HCl (pH 8.4), 100 μM (A)_(n), 100 μM(dT)₂₀, and either 40 mM KCl, 6 mM MgCl₂, 10 mM dithiothreitol, and 500μM [³H]dTTP (85 cpm/pmole), or 100 mM KCl, 1 mM MnCl₂, and 200 μM[³H]dTTP (92 cpm/pmole). Reaction mixtures (50 μl) with(C)_(n):(dG)₁₂₋₁₈ contained 50 mM Tris-HCl (pH 8.4), 60 μM (C)_(n), 24μM (dG)₁₂₋₁₈, and either 50 mM KCl, 10 mM MgCl₂, 10 mM dithiothreitol,and 100 μM [³H]dGTP (132 cpm/pmole), or 100 mM KCl, 0.5 mM MnCl₂, and200 μM [³H]dGTP (107 cpm/pmole). Reaction mixtures also contained either2.5 units of the Tth DNA polymerase (Perkin-Elmer) or 2.5 units of theTne DNA polymerase. Incubations were at 45° C. for 10 min followed by75° C. for 20 min.

The table shows the results of determining the relative levels ofincorporation of Tne and Tth DNA polymerase with (A)_(n):(dT)₂₀ and(C)_(n):(dG)₁₂₋₁₈ in the presence of Mg⁺⁺ and Mn⁺⁺. Tne DNA polymeraseappears to be a better reverse transcriptase than Tth DNA polymeraseunder reaction conditions more specific for reverse transcriptase, i.e.,in the presence of (A)_(n):(dT)₂₀ with Mg⁺⁺ and (C)_(n):(dG)₁₂₋₁₈ withMn⁺⁺ or Mg⁺⁺.

DNA Polymerase Activity of Tth and Tne DNA Polymerase with(A)_(n):(dT)₂₀ and (C)_(n):(dG)₁₂₋₁₈ DNA Polymerase Activity (pMolesComplementary [³H]dNTP Incorporated) (A)_(n):(dT)₂₀ (C)_(n):(dG) EnzymeMg⁺⁺ Mn⁺⁺ Mg⁺⁺ Mn⁺⁺ Tne 161.8 188.7 0.6 4.2 Tth 44.8 541.8 0 0.9

Example 9 Construction of Thermotoga neapolitana 3′-to-5′ ExonucleaseMutant

The amino acid sequence of portions of the Tne DNA polymerase wascompared with other known DNA polymerases such as E. coli DNA polymerase1, Taq DNA polymerase, T5 DNA polymerase, and T7 DNA polymerase tolocalize the regions of 3′-to-5′ exonuclease activity, and the dNTPbinding domains within the DNA polymerase. One of the 3′-to-5′exonuclease domains was determined based on the comparison of the aminoacid sequences of various DNA polymerases (Blanco, L., et al. Gene 112:139-144 (1992); Braithwaite and Ito, Nucleic Acids Res. 21: 787-802(1993)) is as follows:

Tne 318 PSFALD*LETSS 328 (SEQ ID NO:4) Pol I 350 PVFAFDTETDS 360 (SEQ IDNO:5; Braithwaite and Ito, supra) T5 133 GPVAFDSETSA 143 (SEQ ID NO:6;Braithwaite and Ito, supra) T7 1 MIVSDIEANA 10 (SEQ ID NO:7; Braithwaiteand Ito, supra).

As a first step to make the Tne DNA polymerase devoid of 3′→5′exonuclease activity, a 2 kb Sph fragment from pSport-Tne was clonedinto M13mp19 (LTI, Gaithersburg, Md.). The recombinant clone wasselected in E. coli DH5αF′IQ (LTI, Gaithersburg, Md.). One of the cloneswith the proper insert was used to isolate uracilated single-strandedDNA by infecting E. coli CJ236 (Biorad, California) with the phageparticle obtained from E. coli DH5αF′IQ. An oligonucleotide, GA CGT TTCAAG CGC TAG GGC AAA AGA (SEQ ID NO:8) was used to perform site directedmutagenesis. This site-directed mutagenesis converted AsP³²³ (indicatedas * above) to Ala³²³. An Eco47III restriction site was created as partof this mutagenesis to facilitate screening of the mutant followingmutagenesis. The mutagenesis was performed using a protocol as describedin the Biorad manual (1987) except T7 DNA polymerase was used instead ofT4 DNA polymerase (USB, Cleveland, Ohio). The mutant clones werescreened for the Eco47III restriction site that was created in themutagenic oligonucleotide. One of the mutants having the createdEco47III restriction site was used for further study. The mutationAsP³²³ to Ala³²³ has been confirmed by DNA sequencing.

To incorporate the 3′-to-5′ exonuclease mutation in an expressionvector, the mutant phage was digested with SphI and HindIII. A 2 kbfragment containing the mutation was isolated. This fragment was clonedin pUC-Tne to replace the wild type fragment. See FIG. 6A. The desiredclone, pUC-Tne (3′→5), was isolated. The presence of the mutant sequencewas confirmed by the presence of the unique Eco47III site. The plasmidwas then digested with SstI and HindIII. The entire mutant polymerasegene (2.6 kb) was purified and cloned into SstI and HindIII digestedpTrc99 expression vector (Pharmacia, Sweden). The clones were selectedin DH10B (LTI, Gaithersburg, Md.). The resulting plasmid was designatedpTrcTne35. See FIG. 6B. This clone produced active heat stable DNApolymerase.

Example 10 Phenylalanine to Tyrosine Mutant

As discussed supra, the polymerase active site including the dNTPbinding domain is usually present at the carboxyl terminal region of thepolymerase. The sequence of the Tne polymerase gene suggests that theamino acids that presumably contact and interact with the dNTPs arepresent within the 694 bases starting at the internal BamHI site. SeeFIG. 4 and FIGS. 5A and 5B. This conclusion is based on homology with aprototype polymerase E. coli DNA polymerase 1. See Polisky et al., J.Biol. Chem. 265:14579-14591 (1990). The sequence of the carboxylterminal portion of the polymerase gene is shown in FIGS. 5A and 5B.Based upon this sequence, it is possible to compare the amino acidsequence within the O-helix for various polymerases. The completesequence of the DNA polymerase is shown in SEQ ID NO:3. Thecorresponding O-helix region band on the sequence in FIGS. 5A and 5Bincludes amino acids 59 to 72.

Tne 722 RRVGKMVNFSIIYG 735 (SEQ ID NO:9) Pol I 754 RRSAKAINFGLIYG 767(SEQ ID NO:10) T5 562 RQAAKAITFGILYG 575 (SEQ ID NO:11) T7 518RDNAKTFIYGFLYG 531 (SEQ ID NO:12) Taq 659 RRAAKTINFGVLYG 672 (SEQ IDNO:13)

It was shown that by replacing the phenylalanine residue of Taq DNApolymerase, the polymerase becomes non-discriminating againstnon-natural nucleotides such as dideoxynucleotides. See application Ser.No. 08/525,087 entitled “Mutant DNA Polymerases and Use Thereof” of DebK. Chatterjee, filed Sep. 8, 1995, specifically incorporated herein byreference. The mutation was based on the assumption that T7 DNApolymerase contains a tyrosine residue in place of the phenylalanine,and T7 DNA polymerase is non-discriminating against dideoxynucleotides.The corresponding residue, Phe⁷⁶² of E. coli PolI is an amino acid thatdirectly interacts with nucleotides. (Joyce and Steitz, Ann. Rev.Biochem. 63:777-822 (1994); Astake, M. J., J. Biol. Chem. 270:1945-1954(1995)). A similar mutant of Tne DNA polymerase was prepared.

In order to change Phe⁷³⁰ of the Tne polymerase to a Tyr⁷³⁰ as numberedin SEQ ID NO:3, site directed mutagenesis was performed using theoligonucleotide GTA TAT TAT AGA GTA GTT AAC CAT CTT TCC A. (SEQ IDNO:14). As part of this oligonucleotide directed mutagenesis, a HpaIrestriction site was created in order to screen mutants easily. The sameuracilated single-stranded DNA and mutagenesis procedure described inExample 9 were used for this mutagenesis. Following mutagenesis, themutants were screened for the HpaI site. Mutants with the desired HpaIsite were used for further study. The mutation has been confirmed by DNAsequencing.

The Phe⁷³⁰ to Tyr⁷³⁰ mutation was incorporated into pUC-Tne by replacingthe wild type SphI-HindIII fragment with the mutant fragment obtainedfrom the mutant phage DNA. The presence of the desired clone, pUC-TneFY,was confirmed by the presence of the unique HpaI site, see FIG. 6A. Theentire mutant polymerase gene was subcloned into pTrc99 as anSstI-HindIII fragment as described above in DH10B. The resulting plasmidwas designated pTrcTneFY. (FIG. 6B). The clone produced active heatstable polymerase.

Example 11 3′-to-5′ Exonuclease and Phe⁷³⁰→Tyr⁷³⁰ Double Mutants

In order to introduce the 3′→5′ exonuclease mutation and thePhe⁷³⁰→Tyr⁷³⁰ mutation in the same expression vector, pTrc99, it wasnecessary to first reconstitute both mutations in the pUC-Tne clone. SeeFIG. 7. Both the pUC-Tne (3′→5′) and the pUC-TneFY were digested withBamHI. The digested pUC-Tne (3′→5′) was dephosphorylated to avoidrecirculation in the following ligations. The resulting fragments werepurified on a 1% agarose gel. The largest BamHI fragment (4.4 kb) waspurified from pUC-Tne (3′→5′) digested DNA and the smallest BamHIfragment (0.8 kb) containing the Phe⁷³⁰→Tyr⁷³⁰ mutation was purified andligated to generate pUC-Tne35FY. The proper orientation and the presenceof both mutations in the same plasmid was confirmed by Eco47III, HpaI,and SphI-HindIII restriction digests. See FIG. 7.

The entire polymerase containing both mutations was subcloned as aSstI-HindIII fragment in pTrc99 to generate pTrcTne35FY in DH10B. Theclone produced active heat stable polymerase.

Example 12 3′-to-5′ Exonuclease, 5′-to-3′ Exonuclease, and Phe⁷³⁰→Tyr⁷³⁰Triple Mutants

In most of the known polymerases, the 5′-to-3′ exonuclease activity ispresent at the amino terminal region of the polymerase (Ollis, D. L., etal., Nature 313, 762-766, 1985; Freemont, P. S., et al., Proteins 1,66-73, 1986; Joyce, C. M., Curr. Opin. Struct. Biol. 1: 123-129 (1991).There are some conserved amino acids that are implicated to beresponsible for 5′-to-3′ exonuclease activity (Gutman and Minton, Nucl.Acids Res. 21, 4406-4407, 1993). See supra. It is known that 5′-to-3′exonuclease domain is dispensable. The best known example is the Klenowfragment of E. coli Pol I. The Klenow fragment is a natural proteolyticfragment devoid of 5′-to-3′ exonuclease activity (Joyce, C. M., et al.,J. Biol. Chem. 257, 1958-1964, 1990). In order to generate an equivalentmutant for Tne DNA polymerase devoid of 5′-to-3′ exonuclease activity,the presence of a unique SphI site present 680 bases from the SstI sitewas exploited. pUC-Tne35FY was digested with HindIII, filled-in withKlenow fragment to generate a blunt-end, and digested with SphI. The 1.9kb fragment was cloned into an expression vector pTTQ19 (Stark, M. J.R., Gene 51, 255-267, 1987) at the SphI-SmaI sites and was introducedinto DH10B. This cloning strategy generated an in-frame polymerase clonewith an initiation codon for methionine from the vector. The resultingclone is devoid of 219 amino terminal amino acids of Tne DNA polymerase.This clone is designated as pTTQTne535FY. The clone produced active heatstable polymerase. No exonuclease activity could be detected in themutant polymerase as evidenced by lack of presence of unusual sequenceladders in the sequencing reaction. This particular mutant polymerase ishighly suitable for DNA sequencing.

Example 13 5′-to-3′ Exonuclease Deletion and Phe⁷³⁰ →Tyr⁷³⁰ SubstitutionMutant

In order to generate the 5′→3′ exonuclease deletion mutant of the TneDNA polymerase Phe⁷³⁰→Tyr¹³⁰ mutant, the 1.8 kb SphI-SpeI fragment ofpTTQTne535FY was replaced with the identical fragment of pUC-Tne FY. SeeFIG. 8. A resulting clone, pTTQTne5FY, produced active heat stable DNApolymerase. As measured by the rate of degradation of a labeled primer,this mutant has a modulated, low but detectable, 3′→5′ exonucleaseactivity compared to wild type Tne DNA polymerase. M13/pUC Forward23-Base Sequencing Primer™, obtainable from LTI, Gaithersburg, Md., waslabeled at the 5′ end with [P³²] ATP and T4 kinase, also obtainable fromLTI, Gaithersburg, Md., as described by the manufacturer. The reactionmixtures contained 20 units of either wild-type or mutant Tne DNApolymerase, 0.25 pmol of labeled primer, 20 mM tricine, pH 8.7, 85 mMpotassium acetate, 1.2 mM magnesium acetate, and 8% glycerol. Incubationwas carried out at 70° C. At various time points, 10 μl aliquots wereremoved to 5 μl cycle sequencing stop solution and were resolved in a 6%polyacrylamide sequencing gel followed by andoradiography. While thewild-type polymerase degraded the primer in 5 to 15 minutes, it took themutant polymerase more than 60 minutes for the same amount ofdegradation of the primer. Preliminary results suggest that this mutantpolymerase is able to amplify more than 12 kb of genomic DNA when usedin conjunction with Taq DNA polymerase. Thus, the mutant polymerase issuitable for large fragment PCR.

Example 14 Purification of the Mutant Polymerases

The purification of the mutant polymerases was done essentially asdescribed in U.S. patent application Ser. No. 08/370,190, filed Jan. 9,1995, entitled “Cloned DNA Polymerases for Thermotoga neapolitana,” andas in Example 6, supra, with minor modifications. Specifically, 5 to 10grams of cells expressing cloned mutant Tne DNA polymerase were lysed bysonication with a Heat Systems Ultrasonic, Inc. Model 375 machine in asonication buffer comprising 50 mM Tris-HCl (pH 7.4); 8% glycerol; 5 mM2-mercaptoethanol, 10 mM NaCl, 1 mM EDTA, and 0.5 mM PMSF. Thesonication sample was heated at 75° C. for 15 minutes. Following heattreatment, 200 mM NaCl and 0.4% PEI was added to remove nucleic acids.The extract was centrifuged for clarification. Ammonium sulfate wasadded to 48%, the pellet was resuspended in a column buffer consistingof 25 mM Tris-HCl (pH 7.4); 8% glycerol; 0.5% EDTA; 5 mM2-mercaptoethanol; 10 mM KCl and loaded on a heparin agarose (LTI)column. The column was washed with 10 column volumes using the loadingbuffer and eluted with a 10 column volume buffer gradient from 10 mM to1 M KCl. Fractions containing polymerase activity were pooled anddialyzed in column buffer as above with the pH adjusted to 7.8. Thedialyzed pool of fractions were loaded onto a MonoQ (Pharmacia) column.The column was washed and eluted as described above for the heparincolumn. The active fractions are pooled and a unit assay was performed.

The unit assay reaction mixture contained 25 mM TAPS (pH 9.3), 2 mMMgCl₂, 50 mM KCl, 1 mM DTT, 0.2 mM dNTPs, 500 μg/ml DNAse I treatedsalmon sperm DNA, 21 mCi/ml [αP³²] dCTP and various amounts ofpolymerase in a final volume of 50 μl. After 10 minutes incubation at70° C., 10 μl of 0.5 M EDTA was added to the tube. TCA precipitablecounts were measured in GF/C filters using 40 μl of the reactionmixture.

Example 15 DNA Sequencing with the Mutant Polymerases

M13/pUC 23-base forward sequencing primer was ³²P-end-labeled for use insequencing by incubating the following mixture at 37° C. for 10 minutes:60 mM Tris-HCl (pH 7.8), 10 mM MgCl₂, 200 mM KCl, 0.2 μM primer, 0.4 μM(2 μCi/μl) [γ-³²P]ATP, 0.2 U/μl T4 polynucleotide kinase. Labeling wasterminated by incubating at 55° C. for 5 minutes.

Four 10 μl base-specific sequencing reactions were set up for each test.The polymerase and the ddNTP concentrations were varied as follows:

Tne DNA Test polymerase [ddATP] [ddCTP] [ddGTP] [ddTTP] 1 wild-type 0.4mM 0.2 mM 0.04 mM 0.4 mM 2 TneFY 0.4 mM 0.2 mM 0.04 mM 0.4 mM 3 TneFY0.04 mM 0.02 mM 0.004 mM 0.04 mM 4 TneFY 0.004 mM 0.002 mM 0.0004 mM0.004 mM 5 Tne35FY 0.4 mM 0.2 mM 0.04 mM 0.4 mM 6 Tne35FY 0.04 mM 0.02mM 0.004 mM 0.04 mM 7 Tne35FY 0.004 mM 0.002 mM 0.0004 mM 0.004 mM 8Tne535FY 0.4 mM 0.2 mM 0.04 mM 0.4 mM 9 Tne535FY 0.04 mM 0.02 mM 0.004mM 0.04 mM 10 Tne535FY 0.004 mM 0.002 mM 0.0004 mM 0.004 mM

Other components of the reaction were held constant: 1.1 nM pUC 18 DNA,22 nM ³²P-end-labeled primer, 30 mM Tris-HCl (pH 9.0), 5 mM MgCl₂, 50 mMKCl, 0.05% (w/v) W-1, 0.056 U/μl DNA polymerase (see Table), 20 μM dATP,20 μM dCTP, 20 μM 7-deaza-dGTP, 20 μM dTTP. Samples were incubated in athermal cycler at 95° C. for 3 minutes, followed by 20 cycles of (30seconds at 95° C., 30 seconds at 55° C., 60 seconds at 70° C.) and 10cycles of (30 seconds at 95° C., 60 seconds at 70° C.). Reactions wereterminated with 5 μl of stop solution (95% (v/v) formamide, 10 mM EDTA(pH 8.0), 0.1% (w/v) bromophenol blue, 0.1% (w/v) xylene cyanol anddenatured for two minutes at 70° C. Three μl aliquots were separated ona 6% TBE/urea sequencing gel. The dried gel was exposed to BioMAX-MRx-ray film for 16 hours.

Results

Cycle sequencing reactions using P³² end-labeled primers were preparedusing wild-type Tne DNA polymerase and each of the three mutants, TneFY,Tne35FY, and Tne535FY. All four of the polymerases produced sequencingladders. The TneFY mutant gave only a 9 base sequencing ladder when theTaq cycle sequencing reaction conditions were used. This is suggestiveof premature termination due to efficient ddNTP incorporation. Dilutingthe dideoxynucleotides by a factor of 100 extended the ladder to about200 bases The F→Y mutation in the TneFY polymerase therefore alloweddideoxynucleotides to be incorporated at a much higher frequency thanfor wild-type polymerase. The Tne35FY mutant demonstrated a similarability to incorporate dideoxynucleotides. In this case, the sequenceextended to beyond 400 bases and the excess P³² end-labeled M13/pUCforward 23-Base sequencing primer band remained at the 23-base positionin the ladder. The persistence of the 23-base primer band confirmed thatthe 3′→5′ exonuclease activity had been significantly reduced. TheTne535FY mutant performed similarly to the Tne35FY mutant except thatthe signal intensity increased by at least fivefold. The background wasvery low and relative band intensities were extremely even, showing nopatterns of sequence-dependent intensity variation.

Example 16 Generation of 5′-3′ Exonuclease Mutant of Full Length Tne DNAPolymerase 1. Identification of Two Amino Acids Responsible for 5′→3′Exonuclease Activity

Tne DNA polymerase contains three enzymatic activities similar to E.coli DNA polymerase I: 5′-3′ DNA polymerase activity, 3′-5′ exonucleaseactivity and 5′-3′ exonuclease activity. This example is directed to theelimination of the 5′-3′ exonuclease activity in full length Tne DNApolymerase. Gutman and Minton (Nucleic Acids Res. 1993, 21, 4406-4407)identified six (A-F) conserved 5′-3′ exonuclease domains containing atotal of 10 carboxylates in various DNA polymerases in the polI family.Seven out of 10 carboxylates (in domains A, D and E) have beenimplicated to be involved in divalent metal ions binding as judged fromthe crystal structure (Kim et al. Nature, 1995, 376, 612-616) of Taq DNApolymerase. However, there was no clear demonstration that thesecarboxylates are actually involved 5′-3′exonuclease activity. In orderto find out the biochemical characteristics of some of thesecarboxylates, two of the aspartic acids in domains A and E were chosenfor mutagenesis. The following aspartic acids in these two domains wereidentified:

Tne DNA polymerase: 5 F L F D⁸ G T 10 (domain A)Taq DNA polymerase: 15 L L V D¹⁸ G H 20 andTne DNA polymerase: 132 S L I T G D¹³⁷ K D M L 141 (domain E)Taq DNA polymerase: 137 R I L T A D¹⁴² K D L Y 146

2. Isolation of Single Stranded DNA for Mutagenesis

Single stranded DNA was isolated from pSportTne (see infra). pSportTnewas introduced into DH5αF′IQ (LTI, Gaithersburg, Md.) by transformation.A single colony was grown in 2 ml Circle Grow (Bio 101, CA) medium withampicillin at 37° C. for 16 hrs. A 10 ml fresh media was inoculated with0.1 ml of the culture and grown at 37° C. until the A590 reachedapproximately 0.5. At that time, 0.1 ml of M13KO7 helper phage (1×10¹¹pfu/ml, LTI) was added to the culture. The infected culture was grownfor 75 min. Kanamycin was then added at 50 μg/ml, and the culture wasgrown overnight (16 hrs.). The culture was spun down. 9 ml of thesupernatant was treated with 50 μg each of RNaseA and DNaseI in thepresence of 10 mM MgCl₂ for 30 min. at room temperature. To thismixture, 0.25 volume of a cocktail of 3M ammonium acetate plus 20%polyethylene glycol was added and incubated for 20 min. on ice toprecipitate phage. The phage was recovered by centrifugation. The phagepellet was dissolved in 200 μl of TE (10 mM Tris-HCl (pH 8) and 1 mMEDTA). The phage solution was extracted twice with equal volume ofbuffer saturated phenol (LTI, Gaithersburg, Md.), twice with equalvolume of phenol:chlorofomm:isoamyl alcohol mixture (25:24:1, LTI,Gaithersburg, Md.) and finally, twice with chloroform:isoamyl alcohol(24:1). To the aqueous layer, 0.1 volume of 7.5 M ammonium acetate and2.5 volume of ethanol were added and incubated for 15 min. at roomtemperature to precipitate single stranded DNA. The DNA was recovered bycentrifugation and suspended in 200 μl TE.

3. Mutagenesis of D⁸ and D¹³⁷

Two oligos were designed to mutagenize D⁸ and D¹³⁷ to alanine. Theoligos are: 5′ GTAGGCCAGGGCTGTGCCGGCAAAGAGAAATAGTC 3′ (SEQ ID NO:15)(D8A) and 5′ GAAGCATATCCTTGGCGCCGGTTAT TATGAAAATC 3′ (SEQ ID NO:16)(D137A). In the D8A oligo a NgoAIV (bold underlined) and in the oligoD137A a KasI (bold-underlined) site was created for easy identificationof clones following mutagenesis. 200 pmol of each oligo was kinasedaccording to the Muta-gene protocol (Bio-Rad, CA) using 5 units of T4Kinase (LTI, Gaithersburg, Md.). 200 ng of single stranded DNA wasannealed with 2 pmol of oligo according to the Muta-gene protocol. Thereaction volume was 10 μl. Following the annealing step, complementaryDNA synthesis and ligation was carried out using 5 units of wild-type T7DNA polymerase (USB, Ohio) and 0.5 unit T4 ligase (LTI). 1 μl of thereaction was used to transform a MutS E. coli (obtainable from Dr. PaulModrich at the Duke University, NC) and selected in agar platescontaining ampicillin. A control annealing and synthesis reaction wascarried out without addition of any oligo to determine the background.There were 50-60 fold more colonies in the transformation plates withthe oligos than without any oligo. Six colonies from each mutagenicoligo directed synthesis were grown and checked for respectiverestriction site (NgoAIV or KasI). For D8A (NgoAIV), 4 out of 6generated two fragments (3 kb and 4.1 kb). Since pSportTne has an NgoAIVsite near the f1 intergenic region, the new NgoAIV site within the TneDNA polymerase produced the expected fragments. The plasmid wasdesignated as pSportTneNgoAIV. For D137A (KasI), 5 out of 6 clonesproduced two expected fragments of 1.1 kb and 6 kb in size. SincepSportTne has another KasI site, the newly created KasI site generatedthese two expected fragments. The plasmid was designated aspSportTneKasI. Both D8A and D137A mutations have been confirmed by DNAsequencing.

4. Reconstruction of the Mutant Polymerase into Expression Vector

During the course of expression of Tne DNA polymerase or mutant Tne DNApolymerase, a variety of clones were constructed. One such clone wasdesignated as pTTQ Tne SeqS1. This plasmid was constructed as follows:first, similar to above mutagenesis technique glycine 195 was changed toan aspartic acid in pSportTne. A mutation in the corresponding aminoacid in E. coli DNA polymeraseI (polA214, domain F) was found to havelost the 5′-3′ exonuclease activity (Gutman and Minton, see above). AnSspI site was created in the mutant polymerase. Second, a 650 bpSstI-SphI fragment containing the G195D mutation was subcloned inpUCTne35FY (see infra) to replace the wild type fragment. This plasmidwas called pUCTne3022. Finally, the entire mutant Tne DNA polymerase wassubcloned from pUCTne3022 into pTTQ18 as SstI-HindIII fragment togenerate pTTQTneSeqS1. To introduce the mutation D8A or D137A in thisexpression vector, the 650 bp SstI-SphI was replaced with the sameSstI-SphI fragment from pSportTneNgoAIV or pSportTneKasI. The plasmidswere designated as pTTQTneNgo(D8A) and pTTQTneKas(D137A), respectively.

5. Confirmation of the Mutations by DNA Sequencing

DNA sequencing of both mutant polymerases confirmed the presence of therestriction site NgoAIV as well as the mutation D8A; and KasI site aswell as the mutation D137A. Also confirmed by DNA sequencing was thepresence of the mutation D323A and the Eco47III restriction site in the3′-5′exonuclease region. In addition, confirmed by DNA sequencing wasthe F730Y mutation and the HpaI restriction site in the O-helix regionof the mutant Tne DNA polymerase.

6. 5′-3′Exonuclease Activity of the Mutant Tne DNA Polymerases

The full length mutant DNA polymerase was purified as described above.The 5′-3′exonuclease activity was determined as described in the LTIcatalog. Briefly, 1 pmol of labeled (³²P) HaeIII digested λ DNA (LTI)was used for the assay. The buffer composition is: 25 mM Tris-HCl (pH8.3), 5 mM MgCl₂, 50 mM NaCl, 0.01% gelatin. The reaction was initiatedby the addition of 0, 2, 4, 6 and 10 units of either wild type or mutantTne DNA polymerase in a 50 μl reaction. The reaction mix was incubatedfor 1 hr at 72° C. A 10 μl aliquot was subjected to PEI-cellulose thinlayer chromatography and the label released was quantitated by liquidscintillation. In this assay, both D8A and D137A mutants showed lessthan 0.01% label release compared to the wild type Tne DNA polymerase.The result demonstrates that in both D8A and D137A mutants the5′-3′exonuclease activity has been considerably diminished. Thus, it hasbeen confirmed for the first time that these two aspartates are involvedwith the 5′-3′ exonuclease activity.

7. DNA Sequencing Characteristics of the Mutant DNA Polymerases

Four separate base-specific reactions of the following composition wereset up for each Tne polymerase mutant. 6.5 nM pUC18, 111 nM M13/pUC 23base forward sequencing primer, 30 mM Tris-HCl (pH 9.0), 5 mM MgCl₂, 10mM NaCl, 10 mM DTT, 0.05% (w/v) W-1, 0.00185 U/μl inorganicpyrophosphatase, 0.37 μCi/μl (0.37 μM) [α-³⁵S]dATP, 16.7 μM α-thio-dATP,16.7 μM dCTP, 16.7 μM 7-deaza-dGTP, 16.7 μM dTTP, and either 0.042 μMddATP, 0.3 μM ddCTP, 0.255 μM ddGTP or 0.375 μM ddTTP. In thesereactions, the concentrations of the various mutants were: 0.185 U/μlTne535FY, or 0.170 U/μl D8A, or 0.185 U/μl D137A. Reaction volumes were6 μl each. Sample tubes were incubated in an MJ Research DNA Enginethermal cycler at 95° C. for 3 minutes, followed by 20 cycles of (30seconds at 95° C., 30 seconds at 55° C. and 60 seconds at 70° C.), and10 cycles of (30 seconds at 95° C. and 60 seconds at 70° C.). Reactionswere terminated with 3 μl of stop solution (95% (v/v) formamide, 10 mMEDTA (pH 8.0), 0.1% (w/v) bromophenol blue, 0.1% (w/v) xylene cyanol)and denatured for two minutes at 70° C. Three μl aliquots were separatedon a 6% TBE/urea sequencing gel. The dried gel was exposed to KodakBioMAX x-ray film at room temperature approximately 18 hours.

The results of the sequencing data suggest that both D8A and D137Amutants of Tne DNA polymerase produced equivalent sequence ladders withequal band intensity in all 4 lanes comparable to another Tne DNApolymerase where the 5′-exonuclease domain was deleted (Tne535FY). Thisresult also suggests that both D8A and D137A mutants are devoid of5′-exonuclease activity since no false bands are seen in the sequencingladder, a characteristic of 5′-3′ exonuclease containing DNA polymerase.

Example 17 Advantages of Tne DNA Polymerase Mutant in SequencingReactions

In this example, the Tne DNA polymerase of Example 12 was used which hasthe Phe⁷³⁰→Tyr⁷³⁰ mutation (making it non-discriminatory for dNTPs overddNTPs), the AsP³²³→Ala³²³ mutation (which substantially reduces3′-to-5′ exonuclease activity), and the N-terminal 219 amino aciddeletion mutation (which eliminates 5′-to-3′ exonuclease activity).

Sequenase Ver 2.0™ a modified T7 DNA polymerase sold by AmershamInternational plc, Little Chalfont, England.

Taq DNA polymerase was purchase from LTI, Gaithersburg, Md.

Thermo Sequenase™ is a Taq F→Y mutant containing a 5′-exonucleasedeletion sold by Amersham International plc, Little Chalfont, England.

AmpliTaq FS™ is a Taq F→Y mutant believed to contain a Gly³⁷ mutationsold by Perkin Elmer ABI, Foster City, Calif.

Sequitherm™ is a thermophilic DNA polymerase sold by Epicenter, Madison,Wis.

Methods

³⁵S cycle Sequencing with Tne DNA Polymerase

Four separate base-specific reactions of the following composition areset up for each template: 6.5 nM dsDNA, 111 nM primer, 30 mM Tris-HCl(pH 9.0), 5 mM MgCl₂, 10 mM NaCl, 10 mM DTT, 0.05% (w/v) W-1, 0.185 U/μLTne DNA polymerase mutant, 0.00185 U/μl thermophilic inorganicpyrophosphatase, 0.37 μCi/μl (0.37 μM) [α-³⁵S]dATP, 16.7 μM α-thio-dATP,16.7 μM dCTP, 16.7 μM 7-deaza-dGTP, 16.7 μM dTTP, and either 0.042 μMddATP, 0.3 μM ddCTP, 0.255 μM ddGTP or 0.375 μM ddTTP. Reaction volumesare 6 μl each. Sample tubes are incubated in an MJ Research DNA Enginethermal cycler at 95° C. for 3 minutes, followed by 20 cycles of (30seconds at 95° C., 30 seconds at 55° C. and 60 seconds at 70° C.), and10 cycles of (30 seconds at 95° C. and 60 seconds at 70° C.). Reactionsare terminated with 3 μl of stop solution (95% (v/v) formamide, 10 mMEDTA (pH 8.0), 0.1% (w/v) bromophenol blue, 0.1% (w/v) xylene cyanol)and denatured for 2 minutes at 70° C. Three microliter aliquots areseparated on a 6% TBE/urea sequencing gel. The dried gel is exposed toKodak BioMAX x-ray film at room temperature for approximately 18 hours,unless otherwise specified.

³²P-end Labeled Primer Cycle Sequencing with Tne DNA Polymerase

The sequencing primer is labeled by incubating the following 5 μlreaction for 10 minutes at 37° C.: 60 mM Tris-HCl, 10 mM MgCl₂, 200 mMKCl, 0.6 μM primer, 0.4 μM (2 μCi/μl) [γ-³²P]ATP, 0.2 U/μl T4polynucleotide kinase. The reaction is stopped by incubating 5 minutesat 55° C. Four separate base-specific reactions of the followingcomposition are then set up for each template: 1.1 nM dsDNA, 67 nM³²P-end-labeled primer, 30 mM Tris-HCl (pH 9.0), 5 mM MgCl₂, 50 mM KCl,0.05% (w/v) W-1, 0.185 U/μl Tne DNA polymerase, 0.00185 U/μlthermophilic inorganic pyrophosphatase, 20 μM dATP, 20 μM dCTP, 20 μM7-deaza-dGTP, 20 μM dTTP, and either 0.4 μM ddATP, 0.4 μM ddCTP, 0.4 μMddGTP or 0.4 μM ddTTP. Reaction volumes are 10 μl each. Sample tubes areincubated in an MJ Research DNA Engine thermal cycler at 95° C. for 3minutes, followed by 20 cycles of (30 seconds at 95° C., 30 seconds at55° C. and 60 seconds at 70° C.), and 10 cycles of (30 seconds at 95° C.and 60 seconds at 70° C.). Reactions are terminated with 5 μl of stopsolution (95% (v/v) formamide, 10 mM EDTA (pH 8.0), 0.1% (w/v)bromophenol blue, 0.1% (w/v) xylene cyanol) and denatured for 2 minutesat 70° C. Three μl aliquots are separated on a 6% TBE/urea sequencinggel. The dried gel is exposed to Kodak BioMAX x-ray film at roomtemperature for approximately 18 hours, unless otherwise specified.

Single-Extension Sequencing with Tne DNA Polymerase

This reaction requires either ssDNA or denatured dsDNA. The DNA isannealed to primer in a 10 μl volume by heating for five minutes at 50°C. under the following reaction conditions: 150 nM dsDNA and 150 nMprimer or 50 nM ssDNA and 50 nM primer with 60 mM Tris-HCl (pH 9.0), 60mM KCl, 10 mM MgCl₂, 0.1% (w/v) W-1. The following labeling reaction isthen incubated for five minutes at 50° C. in a 15.5 μl volume: 10 μLannealed DNA-primer 0.32 μCi/μl (0.32 μM) [α-³⁵S]dATP, 48.4 mM Tris HCl(pH 9.0), 48.4 mM KCl, 8.1 mM MgCl₂, 194 nM dCTP, 194 nM 7-deaza-dGTP,194 nM dTTP, 6.5 nM DTT, 0.081% (w/v) W-1, 0.32 U/μl Tne DNA polymerase,0.0032 U/μl thermophilic inorganic pyrophosphatase. The label mixture isthen dispensed into four base-specific reaction tubes. Each tubecontains a total reaction volume of 6 μl and is incubated for 5 minutesat 70° C. under the following conditions: DNA-labeled primer 0.19 μCi/μl(0.19 μM) [α-³⁵S]dATP, 28 mM Tris-HCl (pH 9.0), 28 mM KCl, 4.7 mM MgCl₂,42 μM dATP, 42 μM dCTP, 42 μM 7-deaza-dGTP, 42 μM dTTP, 3.8 mM DTT,0.047% (w/v) W-1, 0.19 U/μl Tne DNA polymerase, 0.0019 U/μl thermophilicinorganic pyrophosphatase and either 0.83 μM ddATP, 0.83 μM ddCTP, 0.83μM ddGTP or 0.83 μM ddTTP. Reactions are terminated by adding 4 μl ofstop solution (95% (v/v) formamide, 10 mM EDTA (pH 8.0), 0.1% (w/v)bromophenol blue, 0.1% (w/v) xylene cyanol) and denatured for 2 minutesat 70° C. Two μl aliquots are separated on a 6% TBE/urea sequencing gel.The dried gel is exposed to Kodak BioMAX x-ray film at room temperaturefor approximately 2 hours, unless otherwise specified.

Fluorescent Dye Primer Sequencing with Tne DNA Polymerase

Four base-specific reactions are set up for each template. The A and Creaction volumes are 5 μl and the G and T reaction volumes are 10 μl.The composition of the reactions are as follows: 20 nM dsDNA or 10 nMssDNA, with 30 mM Tris-HCl (pH 9.0), 30 mM KCl, 5 mM MgCl₂, 0.05% (w/v)W-1, 20 μM dATP, 20 μM dCTP, 20 μM 7-deaza-dGTP, 20 μM dTTP, 0.29 U/μlTne DNA polymerase, 0.0029 U/μl thermophilic inorganic pyrophosphatase.Each of the four tubes also contains a base-specific dye primer andddNTP as follows:

A: 0.4 μM JOE dye primer, 0.4 μM ddATP

C, 0.4 μM FAM dye primer, 0.4 μM ddCTP

G: 0.4 μM TAMRA dye primer, 0.4 μM ddGTP

T: 0.4 μM ROX dye primer, 0.4 μM ddTTP

Sample tubes are incubated in a thermal cycler at 95° C. for 3 minutes,followed by 20 cycles of (30 seconds at 95° C., 30 seconds at 55° C. and60 seconds at 70° C.), and 10 cycles of (30 seconds at 95° C. and 60seconds at 70° C.). Reactions are pooled, purified over a CentriSep spincolumn, and dried. The dried pellet is dissolved in 3 μl of 83%formamide, 4.2 mM EDTA (pH 8.0) and heated for 2 minutes at 90° C. justbefore loading the entire sample on a 4.75% polyacrylamide/TBE/urea gelin an ABI 373 Stretch machine. The gel is run at 32 watts for 14 hours.Fluorescent Dye Terminator Sequencing with Tne DNA Polymerase

One 20 μl reaction is set up for each template. The composition of thereaction is an follows: 12.5 nM dsDNA or 6.25 nM ssDNA, with 0.16 μMprimer, 30 mM Tris-HCl (pH 9.0), 30 mM KCl, 5 mM MgCl₂, 0.05% (w/v) W-1,0.6 mM dATP, 0.6 mM dCTP, 1.8 mM dITP, 0.6 mM dTTP, 0.5 U/ml Tne DNApolymerase, 0.005 U/μl thermophilic inorganic pyrophosphatase. Thereaction also includes four base-specific dye terminators at a finalconcentration 16-fold lower than the original concentration supplied byABI. The sample tube is incubated in a thermal cycler for 25 cycles of(30 seconds at 96° C., 15 seconds at 50° C. and 4 minutes at 60° C.).The reaction is purified over a CentriSep spin column, and dried. Thedried pellet is dissolved in 3 μl of 83% formamide, 4.2 mM EDTA (pH 8.0)and heated for 2 minutes at 90° C. just before loading the entire sampleon a 4-75% polyacrylamide/TBE/urea gel in an ABI 373 Stretch machine.The gel is run at 32 watts for 14 hours.

Results

Single-Extension Sequencing

FIG. 9 shows that the efficient ³⁵S incorporation by Tne DNA polymerasemutant provides strong signals in single- and double-strand DNAsequencing. Alkali-denatured pUC19 DNA (1.5 pmol) was sequenced usingsingle-extension sequencing with Tne DNA polymerase of Example 12 asdescribed above (set A); film was exposed for only 2 hours. M13 mp19(+)DNA was used at one-tenth the normal amount of template (40 pmol) in theTne DNA polymerase single-extension sequencing reactions as described(set B); film exposed for 20 hours. Since the Tne mutant produces such astrong signal, templates can be used more economically withoutsacrificing sequence quality.

FIG. 10 shows that the Tne DNA polymerase mutant generates clearsequence from plasmids containing cDNAs with poly(dA) tails.Alkali-denatured plasmid DNAs containing cDNA inserts (1.5 pmol) weresequenced using either the Tne DNA polymerase mutant in single-extensionsequencing (sets A and B) as described, or Sequenase Ver 2.0° (set C)following the standard kit protocol. Set A, β-actin cDNA; set B, RPA1cDNA (a replication protein); and set C, RPA2 cDNA (a replicationprotein).

FIG. 11 compares the Tne DNA polymerase mutant, Sequenase™ and Taq DNApolymerase generated sequences from a plasmid containing poly(dC).Plasmid DNA (1.5 pmol) containing a poly(dC)-tailed 5′ RACE-derivedinsert was alkali denatured. The DNA was sequenced using Tne DNApolymerase mutant in single-extension sequencing (set A) as described,Sequenase Ver 2.01 (set B) as described in the kit manual, and by TaqDNA polymerase (set C) following the recommended protocol in theTaqTrack kit (Promega, Madison, Wis.).

Cycle Sequencing

FIG. 12 shows that the Tne DNA polymerase mutant in cycle sequencingproduces ³⁵S-labeled sequence 3-fold stronger than Thermo Sequenase™ andwithout the 60-cycle labeling step. Plasmid DNA (0.5 μg) containing apoly(dC)-tailed 5′ RACE-derived insert was cycled sequenced using TneDNA polymerase mutant (set A) as described; film exposure was 6 hours.Using Thermo Sequenase™ as described in the kit manual, the plasmid DNA(0.5 μg) was labeled with ³⁵S by partial primer extension using anincubation of 60 cycles. This was followed by the standard cyclesequencing protocol in the presence of chain terminators (set B); filmexposure was 18 hours. The plasmid DNA (0.5 μg) was cycle sequencedusing Taq DNA polymerase (set C) as described in the fmol kit manual;film exposure was 18 hours. Note, uneven band intensities in set C.

FIG. 13 shows that the Tne DNA polymerase mutant produces high qualitysequences of in vitro amplified DNA. Templates were in vitro amplifieddirectly from E. coli chromosomal DNA, from plasmid pSC101 and fromhuman genomic DNA, purified by simple isopropanol precipitation andquantitated. DNAs (100 fmol) were cycle sequenced as described using theTne DNA polymerase mutant and one of the amplification primers. Set A,E. coli β polI (450 bp); set B, E. coli rrsE (˜350 bp); set C, ori frompSC101 (˜1.5 kb); and set D, an exon from human HSINF gene (˜750 bp);amplified product sizes in parentheses. Note, these DNAs could not besequenced using Thermo Sequenase™ because the primers did not meet theextra requirements for the labeling reaction.

FIGS. 14A and 14B show that the Tne DNA polymerase mutant providessuperior sequence from double-stranded DNA clones containing poly(dA) orpoly(dC) stretches. FIG. 14A, supercoiled plasmid DNAs containinginserts with homopolymers were cycle sequenced using the Tne DNApolymerase mutant as described; film exposure was 6 hours. Set A, RPA1;set B, elf (cap binding protein); and set C, a poly(dC)-tailed 5′RACE-derived insert.

FIG. 14B, supercoiled plasmid DNAs containing inserts with homopolymerswere cycled sequenced using Taq DNA polymerase (set D) in the fmol kitmanual, or SequiTherm™ (sets E-G) following the kit manual; filmexposure was 18 hours. Set D, RPA; set E, RPA; set F, a poly(dC)-tailed5′ RACE-derived insert; and set G, elf. Note, the many false stops,especially in the homopolymer region.

FIG. 15 shows cycle sequencing using the Tne DNA polymerase mutant and³²P end-labeled primer. A sequencing primer was first 5′-end labeledwith ³²P using T4 kinase. A supercoiled plasmid DNA (50 fmol) was cyclesequenced using the Tne DNA polymerase mutant as described; filmexposure was 18 hours. The left and right sets are aliquots of the samereaction, the right set loaded on the gel 45 minutes after the left.

Fluorescent Automated Sequencing

FIGS. 16A-16C and 16D-16F show a comparison of the Tne DNA polymerasemutant (16A-16C) to AmpliTaq FS™ (16D-16F) in fluorescent dye primersequencing. pUC19 DNA was sequenced with dye primers (ABI, Foster City,Calif.) using either the Tne DNA polymerase mutant or AmpliTaq FS™ asdescribed.

FIGS. 17A-17C and 17D-17F show a comparison of the Tne DNA polymerasemutant (17A-17C) to AmpliTaq FS™ (17D-17F) in fluorescent dye terminatorsequencing. pUC19 DNA was sequenced with dye terminators (ABI, FosterCity, Calif.) using either the Tne DNA polymerase mutant or AmpliTaq FS™as described. Note, greater evenness of peak heights with Tne.

These results demonstrate that the Tne DNA polymerase mutant givesunexpectedly better results in DNA sequencing compared to other DNApolymerases, whether they are similar mutants or not.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated by referencein their entirety.

1. A substantially pure Thermotoga neapolitana (Tne) DNA polymerase. 2.The DNA polymerase of claim 1, which is isolated from Thermotoganeapolitana.
 3. The DNA polymerase of claim 2, which is isolated fromThermotoga neapolitana DSM 5068 and said DNA polymerase has a molecularweight of about 100 kilodaltons.
 4. The DNA polymerase of claim 1 havingthe amino acid sequence of SEQ ID NO:3.
 5. A Tne DNA polymerase mutantwhich is modified at least one way selected from the group consisting of(a) to reduce or eliminate the 3′→5′ exonuclease activity of thepolymerase; (b) to reduce or eliminate the 5′→3′ exonuclease activity ofthe polymerase; and (c) to reduce or eliminate discriminatory behavioragainst a dideoxynucleotide.
 6. The DNA polymerase mutant of claim 5,which is modified at least two ways.
 7. The DNA polymerase mutant ofclaim 5, which is modified three ways.
 8. The Tne DNA polymerase mutantof claim 5 which comprises a mutation in the O-helix of said DNApolymerase resulting in said DNA polymerase becoming non-discriminatingagainst dideoxynucleotides.
 9. The DNA polymerase of claim 8, whereinsaid O-helix is defined as RXXXKXXXFXXXYX, wherein X is any amino acid.10. The Tne DNA polymerase as claimed in claim 10, wherein said mutationis a Phe⁷³⁰→Tyr⁷³⁰ substitution.
 11. The Tne DNA polymerase of claim 5,wherein said DNA polymerase is a Tne DNA polymerase having substantiallyreduced 3′→5′ exonuclease activity.
 12. The mutant Tne DNA polymerase asclaimed in claim 11, wherein said mutant is a AsP³²³→Ala³²³substitution.
 13. The mutant Tne DNA polymerase as claimed in claim 5,wherein said mutant polymerase comprises both a Phe⁷³⁰→Tyr⁷³⁰substitution and a AsP³²³→Ala³²³ substitution.
 14. The mutant DNApolymerase mutant of claim 5, wherein said DNA polymerase is a Tne DNApolymerase having substantially reduced 5′→3′ exonuclease activity. 15.The mutant Tne DNA polymerase as claimed in claim 14, wherein saidmutant polymerase has a deletion mutation in the N-terminal 5′→3′exonuclease domain.
 16. The mutant Tne DNA polymerase as claimed inclaim 15, wherein said mutant polymerase is devoid of the 219 N-terminalamino acids.
 17. A vector comprising a gene encoding the DNA polymeraseof any one of claims 1 or
 5. 18. The vector of claim 17, wherein saidgene is operably linked to a promoter.
 19. The vector of claim 18,wherein said promoter is selected from the group consisting of a λ-P_(L)promoter, a tac promoter, a trp promoter, and a trc promoter.
 20. A hostcell comprising the vector of claim
 17. 21. A method of producing a DNApolymerase, said method comprising: (a) culturing the host cell of claim20; (b) expressing said gene; and (c) isolating said DNA polymerase fromsaid host cell.
 22. The method of claim 21, wherein said host cell is E.coli.
 23. A method of synthesizing a double-stranded DNA moleculecomprising: (a) hybridizing a primer to a first DNA molecule; and (b)incubating said DNA molecule of step (a) in the presence of one or moredeoxy- or dideoxyribonucleoside triphosphates and the DNA polymerase ofany one of claims 1 or 5, under conditions sufficient to synthesize asecond DNA molecule complementary to all or a portion of said first DNAmolecule.
 24. The method of claim 21, wherein said deoxy- ordideoxyribonucleoside triphosphates are selected from the groupconsisting of dATP, dCTP, dGTP, dTTP, dITP, 7-deaza-dGTP, dUTP, ddATP,ddCTP, ddGTP, ddlTP, ddTTP, [α-S]dATP, [α-S]dTTP, [α-S]dGTP, and[α-S]dCTP.
 25. The method of claim 23, wherein one or more of saiddeoxy- or dideoxyribonucleoside triphosphates are detectably labeled.(a) A method of sequencing a DNA molecule, comprising: (a) hybridizing aprimer to a first DNA molecule; (b) contacting said DNA molecule of step(a) with deoxyribonucleoside triphosphates, the DNA polymerase of anyone of claims 1 or 5, and a terminator nucleotide; (c) incubating themixture of step (b) under conditions sufficient to synthesize a randompopulation of DNA molecules complementary to said first DNA molecule,wherein said synthesized DNA molecules are shorter in length than saidfirst DNA molecule and wherein said synthesized DNA molecules comprise aterminator nucleotide at their 5′ termini; and (d) separating saidsynthesized DNA molecules by size so that at least a part of thenucleotide sequence of said first DNA molecule can be determined. 26.The method of claim 26, wherein said deoxyribonucleoside triphosphatesare selected from the group consisting of dATP, dCTP, dGTP, dTTP, dITP,7-deaza-dGTP, dUTP, [α-S]dATP, [α-S]dTTP, [α-S]dGTP, and [α-S]dCTP. 27.The method of claim 26, wherein said terminator nucleotide is ddTTP,ddATP, ddGTP, ddITP or ddCTP.
 28. The method of claim 26, wherein one ormore of said deoxyribonucleoside triphosphates is detectably labeled.29. The method of claim 26, wherein one or more of said terminatornucleotides is detectably labeled.
 30. A method for amplifying a doublestranded DNA molecule, comprising: (a) providing a first and secondprimer, wherein said first primer is complementary to a sequence at ornear the 3′-termini of the first strand of said DNA molecule and saidsecond primer is complementary to a sequence at or near the 3′-terminiof the second strand of said DNA-molecule; (b) hybridizing said firstprimer to said first strand and said second primer to said second strandin the presence of the DNA polymerase of any one of claims 1 or 5, underconditions such that a third DNA molecule complementary to said firststrand and a fourth DNA molecule complementary to said second strand aresynthesized; (c) denaturing said first and third strand, and said secondand fourth strands; and (d) repeating steps (a) to (c) one or moretimes.
 31. The method of claim 31, wherein said deoxyribonucleosidetriphosphates are selected from the group consisting of dATP, dCTP,dGTP, dTTP, dITP, 7-deaza-dGTP, dUTP, [α-S]dATP, [α-S]dTTP, [α-S]dGTP,and [α-S]dCTP.
 32. A kit for sequencing a DNA molecule, comprising: (a)a first container means comprising the DNA polymerase of any one ofclaims 1 or 5; (b) a second container means comprising one or moredideoxyribonucleoside triphosphates; and (c) a third container meanscomprising one or more deoxyribonucleoside triphosphates.
 33. A kit foramplifying a DNA molecule, comprising: (a) a first container meanscomprising the DNA polymerase of any one of claims 1 or 5; and (b) asecond container means comprising one or more deoxyribonucleosidetriphosphates.
 34. A mutant Tne DNA polymerase having substantiallyreduced or eliminated 5′-3′ exonuclease activity, wherein at least oneof the amino acids corresponding to Asp⁸, Glu¹¹², Asp¹¹⁴, Asp¹¹⁵,Asp¹³⁷, Asp¹³⁹, Gly¹⁰², Gly¹⁸⁷, or Gly¹⁹⁵ has been mutated.
 35. A vectorcoding for the mutant DNA polymerase of claim
 35. 36. A host cellcomprising the vector of claim
 36. 37. A method of producing a mutantTne DNA polymerase having substantially reduced or eliminated 5′-3′exonuclease activity, wherein at least one of the amino acidscorresponding to Asp⁸, Glu¹¹², Asp¹¹⁴, Asp¹¹⁵, Asp¹³⁷, Asp¹³⁹, Gly¹⁰²,Gly¹⁸⁷, or Gly¹⁹⁵ has been mutated, comprising (a) culturing the hostcell of claim 37; (b) expressing the mutant DNA polymerase; and (c)isolating said mutant DNA polymerase.
 38. A method of preparing cDNAfrom mRNA, comprising (a) contacting mRNA with an oligo(dT) primer orother complementary primer to form a hybrid, and (b) contacting saidhybrid formed in step (a) with the Tne DNA polymerase or mutant of claim1 or 5 and dATP, dCTP, dGTP and dTTP, whereby a cDNA-RNA hybrid isobtained.
 39. A method of preparing dsDNA from mRNA, comprising (a)contacting mRNA with an oligo(dT) primer or other complementary primerto form a hybrid, and (b) contacting said hybrid formed in step (a) withthe Tne DNA polymerase or mutant of claim 1 or 5, dATP, dCTP, dGTP anddTTP, and an oligonucleotide or primer which is complementary to thefirst strand cDNA; whereby dsDNA is obtained.